What's going on and welcome to the NBA DFS Q&A show. My name is Tom Vecchio I'll be here for the next 30 minutes taking your questions about tonight's 11 game NBA slate as always This show is every Wednesday 4 p.m. Right here on the Fandle YouTube, Twitch, Twitter and Facebook pages Make sure to like follow subscribe on the give in a platform 11 games on the slate obviously there are a ton of options we can be considering We are just a few days away from the NBA all-star break. There's a lot of value on the slate We have a number of top-tier stars We want to be paying up for the injury news on the slate is You know pretty straightforward in terms of what we're looking at There are a number that we need a number of different players We need to be focusing on LeBron James, John Morant, Morant didn't play last night Van Blee, Anthony Edwards left last night's game early We have Anthony Davis, Liziz, Probable, etc. etc. So pretty straightforward from what we should be seeing some of these are West Coast games So we do need to get some clarity on that You know LeBron James does sit that could put a bit of a priority on Anthony Davis We go over the injuries. We can go over the values that I'm looking at We can go over some of the bets that I have for tonight Grim on YouTube is saying a star quitting Grimes over CJ L.O.B.E. and Justin Holiday Grimes for the Knicks. Yeah, this is I want to say a pretty solid matchup for them I you know I said this on the podcast the daily ice of day The Nets are just not playing defense and you know we have seen What was it there 11 game 10 game losing streak whatever it was like they are not playing defense So I think the Knicks are in a good spot a nice pace of spot for them. I prefer Grimes over L.O.B. I do like L.O.B.E. tonight is simply due to his salary being a little bit cheaper compared to Grimes We have Grimes at 5200 and L.O.B.E. is 4600 for Portland, right? Yeah, he's 4500 and Grimes is 52 so I like them both for different reasons Like I like Grimes because the matchup like this is certainly an awesome spot going up against the Nets We're not playing a whole lot of defense and it's a pace-up spot for the Knicks So we certainly can be going to him. Knicks are 26 and lead Brooklyn's up at nine So push comes to shove. I'm choosing Grimes if you have the salary DJ is asking stack Memphis or Sacramento tonight I'm talking about those games in general because if we're talking about the Sacramento Chicago game I have much more interest in the Chicago side of things In stacking this game, but I think both of them are firmly in play Vucevich is the the covered boy for this thumbnail So I'm a big fan of Vucevich tonight between him DeRosin and Murray these are probably my favorite three stars on tonight's slate And immediately locking them into your lineup leaves you with less than 5000 remaining So if you're talking about stacking those games overall Both great games you want to be attacking 233 over under and a 230 over under We do have a larger spread in Memphis in Portland. We're waiting on the John Morant news I still think that there are plenty of options Whether you're looking at the Anthony Simons on the Portland side of things You know speaking of him. I took the over on his points rebounds assist profits sitting at 30 and a half 29 and a half 30 and a half 30 and a half for Simons that took the over on that So certainly looking to plenty of options in this game and then Chicago and Sacramento This is shaping up to be a very very solid fancy environment Vucevich and DeRosin specifically have been crushing it lately DeRosin going for 35 plus points on the nightly bases Vuce putting up a double double So I absolutely love this matchup on the Sacramento side Where could you be going? You know, so bonus I think makes the natural choice Just because he's arguably their most consistent But I have some interest in going to Harrison Barnes in the midst here Depending on what kind of lineup build you're doing at 6,800 He doesn't fit normally into a stars or scrubs build he falls in this you know mid-range So if he's going to be going over looks I like that and of course DeRion Fox is always a good option If we do see John Morant ruled out, you know, go back to Desmond Bain I think that's certainly an option You know last night wasn't the biggest game for him But the three-point ability especially against a team like Portland will always be there So Memphis, Portland and Sacramento, Chicago Two great games you want to be stacking tonight Speaking of Chicago, I took Chicago minus six tonight This is a very winnable matchup for them They should be taking care of business there one of the best ATS teams this season 34-23 Sacramento surprisingly not that great against the spread And I'm going to Chicago minus six Simons over 30 and a half I also took Kuzma Kyle Kuzma for the Washington Wizards Over his points rebounds assist prop He's kind of the guy now it's 36 and a half I think I might have gotten at 35 He's kind of the guy now for Washington Bradley Beale is done, you know, the Dinwood he has been traded It's a one-point spread against the Indiana Pacers And the Pacers are seemingly always getting in these higher very close game So I'm certainly on board with Kuzma tonight A player that's stepping out and hitting a number of threes this year Is always good Say Goon should be a good low price play with Woodhouse So Wood is listed as doubtful if I remember correctly It's just a he's questionable for tonight So, uh, yeah, Alperine St. Goon for the Rockets would be a very solid play Yeah, we just need a confirmation on Wood He is listed as questionable not officially happen I'm totally on, you know, along the same lines as you on board With Joseph saying that Alperine St. Goon for the Houston Rockets as a bit of a Value play 5100 totally going to him at center slash power forward It is a West Coast game So leave yourself that You know that flexibility like okay if Wood is actually ruled in Where am I going to be pivoting? What am I going to be shifting around? But yes, seeing Goon totally on board when he comes to some value tonight There's a lot of it. Jane McDaniels started Or got or took the place of Anthony Edwards last time I don't know if he actually started the second half I'd have to double check that but McDaniels started or took the minutes over for Anthony I were too left early last night for Minnesota So looking to the value for him if he's going to be in the starting lineup I'm also a fan of going to okay see for Aaron Wayans at $3,600 Taking that value there, you know, I'm kind of big on continuous call will pulpit And I like this match up kind of back and forth There's a few options in this game Like I said, it took the over at Kuzma I kind of like KCP tonight That's also a spot that I'm going for a little bit value $4,200 If you want to lock in Elibi at $4,500 I mean, you're left with $3,600 and you're locking in DeRose and Murray and Vooch And then you have some solid value plays That all should be seeing plenty of minutes that are all in, you know, pretty solid game environments okay sees playing up in pace Portland also playing up in pace back and forth nature between Toronto and Minnesota Beasley or Hart that's a good question Gary's Beasley or Josh Hart Well, that's a that's a real coin flip You know, I might take Josh Hart because I like his ability to fill the statue Not saying that Darius Beasley can't do that but The the pace up spot for Portland in Oklahoma City is obviously very very comparable Portland is 19th in the league. They are playing Memphis who are fifth Okay, see is 17th. They're playing St. Antonio who's sixth. So we're literally talking about like splitting hairs here I would take Josh Hart a little more pure scoring upside. That's just me It's like salt to taste where that saying is like it's literally just personal preference at a certain point. I would lean towards Hart But they're both very solid options obviously new roles for both of them The trades on Portland, Lillard still out, McCollum's gone on OKC We know they're dealing with a number of injuries with SGA still out The door is out, Kenneth Williams is out, etc, etc So there's certainly an opening for both of those players I know that I have exposure to both of them in the first run through of the 25 lineups that I made so far to start That's where I am going with a lot of the place Focusing in on these higher over under games You know this game like Detroit and Boston like this a game I have very very little if any interested There's a chance that There's a chance that we do see some minutes for Peyton Prichard tonight Just because Marcus Smart is listed as doubtful You know as I mentioned on the podcast today They're coming off this huge win last night against Philadelphia They're what is it? They don't play tomorrow night So this means is their last game before the all-star break So it's not going to be a surprise to see Boston kind of take care of business in the first half versus Detroit And then to give ground and Tatum some rest leading into the all-star break And that opens up some minutes for Peyton Prichard potentially So if you're looking for a deeper value play Prichard at 3500 is the option I would go to but With a 213 over under outside of that like I don't have any interest in going to Tatum any interest in going to Brown Not only do we see this with the lowest over under on the slate These are also teams with Boston. They are a heavy under team this season 24 34 and one heavy under team and Detroit is even or slightly less than even 28 28 and one So this doesn't like present a whole lot of scoring upside with this 213 over under So I'm not looking here for basically any options You know, I wouldn't mind taking a shot with Jeremy Grant Maybe City Bay, maybe Kate Cunningham, but I would be sure to You know limit the options in this game To at max one Or or Grant Williams if you're going to go to Grant Williams again at $3,600 for that value Because he got to start last night with Rob Williams listed is out I think that's fine But no more than one player from this game like that is a rule I will set in the optimizer at most one player from this game So between Williams and McDaniels at power forward Lot of value here lot of value here going to Wiggins as well. Like I said, I'm on board with KCP Taking that value there There's this is a great stars and scrub slate if that's what you're looking to do the Rosen Vooch and Murray as my top three As my top three stars There's plenty of value and a lot of the value you can find under $4,000 I like round NATO has a good low price plate too. Yeah round NATO this game against the The basers like this is a pretty solid game environment 225 over under one point spread Like this is everything that we want and you know, both lineups are a little bit thinned out just because of the trades and the injuries, etc etc like When it comes to the wizards, I think KC I think kuzma is like the go-to guy on offense now at least that's what we should be seeing from them KCP providing, you know, plenty of shooting upside for that three-point ability. That's what I'm liking But round NATO out here taking a good amount of a good volume of shots He fills up the stat sheet enough which should allow him to reach or seed fancy value It's not like we're worried about the defense from the Indiana Pacer. So I'm on board with this game I think that the uh, the next game This the next net's game could go slightly overlooked just because it's falling a little bit lower We do have some good value on both sides just because the nets are still without Durant or without kairi Irving I like Quintin Grimes tonight. I like Julius Randlitz night as well We've seen him step up to a new level of production that he really You know didn't have in the beginning portion of season where he kind of regained his form as of late You know Consistently pushing towards a double double or a triple double which he didn't always have in the beginning portion of season He was struggling a little bit and you know, we see this like these lower scoring games were certainly there for him He would certainly reach high in some games, but now we have a higher floor for him So I like Randl personally tonight that might be a spot that I'm willing to go Maybe a little bit more exposure to him compared to the field trying to get a little bit of leverage in torments Always something you want to be doing, you know, if you're confident in a player that is uh You know, you want to be taking a higher stand on him Like when you're right, you want to be the most right about, you know, a player like Randl Who can go over 60 plus and if it's not going to be popular. Well, that's always a you know an added benefit Greatness saying looking at the three by two on the final sportsbook. I'm considering nuggets warriors Sacramento, Chicago, Memphis, Portland for those games. So three by two you're obviously looking for Those three partners the warriors obviously make a lot of sense just because they're gonna have clay and stuff out there That's always good Sacramento, Chicago, Memphis, Portland I would take Memphis, Portland ahead of Sacramento, Chicago Chicago You know, ultimately they don't have a ton of three-point shooters like consistently on their roster We're gonna bring up de rosin's log right here. He doesn't take threes He has two threes over the past three games. He had four three-point attempts against charlie You know, we can have everyone else that's a high for him Like he's a player that is their primary ball hinder and he's one that does not take three pointers So the when he's the primary ball hinder from one of the given teams that he's not willing to take threes or just It's not that he's not willing to take threes. That's just not his game When the ball goes to him getting those early threes like that may not be the case Meanwhile, you look to a team like portland and Memphis if jammer ends out We know desmond bain takes a ton of threes and also anthony simons for portland takes a ton of 16 threes In his most recent game So I would side with the warriors nuggets matchup and Memphis portland for the three by two on the sports book Joseph is asking sugs or grimes. Oh, that's a good question another another one of these tough toss-up of the players in the mid tier So let's see what we're looking at with some projections. Jalen Suggs 32 minutes 30 feint two points is where he should write should be right around and grimes It's right around the same. He's going to be under one feint two point per minute just because the usage is likely going to Julius randall and going to camba. I would take Jalen Suggs in that match in that specific head-to-head for Suggs and grimes, you know this this orlando elante game I think is one that's kind of flying a bit under the radar where you know 226 It looks good, but when you see games that have closer spreads and higher overenders This is a game that's like half a step behind some of the other games where you know, we're looking to okay See for a lot of value where orlando in the grand scheme of things. They're fully healthy Right. This is the lineup that they've been running out there for You know a good portion of the season like there's there's no deep value. Everything's pretty straightforward for both teams Yeah, we have john collins out, but gallinar is going to step in there as he has for the past few games So if this game goes overlooked a little bit, you know, wendell carter is a player that can always go off for Plenty of points treyong has that three point upside. So if you want to get a little bit extra exposure to that game There's nothing wrong with that So the injuries again, if you were attempting to play or you are interested in playing lebron james Be aware Very minor that's thursday's game thursday game all Non-important. So if you're playing lebron james, right? And it's one of the west coast games This is something that you have to obviously have You know pre-plan like where you're going to be going if you have Lebron in your lineups and he gets ruled out the natural pivot would be to have anthony davis Where you have him at power you would have lebron at power forward, you know Make sure that's the case so you can quick swap to anything davis And you have to make sure to leave yourself the 200 dollars So that's the easy way to go about it if you want to you know play lebron Playing him at small forward it is obviously tougher because there's no natural pivot away from him With all these games starting earlier. So i'd make sure to play lebron at power forward if you're again if you're using an optimizer Make sure to select lebron as Power forward only so and you have anything davis as you know an option to swap swap too late Joseph's asking i'm paid up for trade in yokich. So trying to fit in submit in low Low price players. Yeah, totally. You know, we want that scoring outside yokich is always a player that can Have this a ceiling net like no one else can reach for right and and we've seen this over the past few seasons where You know, I like voosevich tonight like he's the coverboy on this On this show for a reason like the matchup for him against sacramento is phenomenal sacramento is second worst in the league against center So i love voosh tonight. He's been unbelievably consistent, but voosh Probably isn't going for 75 points right like that's something that yokich could do like pretty consistently And no one would be too shocked like voosh going for 70 points. That would be a huge game for him He's more realistically in like the 45 to 60 point range. Like that's where we should be seeing voosevich yokich is a player that when you have exposure to him And he goes off for 70 like you're in every contest like you have a chance in every contest So I love yokich tonight. I know i'm gonna have plenty of exposure to him for me It's voosh yokich and then towns in that order when it comes to centers And those are center specific plays not players that have dual position eligibility DJ is asking can I explain the three by two? So this is the three by two. It's one of the I just one of the specials on the fangirl sports book two plus threes in the first three minutes So you're you're saying, you know, will there be Two three pointers at least two three pointers made in the first three minutes of the given game So you can look to parlay them you can say obviously it's a yes or no prop So you can choose which props you are which games have the best lines If you're looking for some of those as uh grim brought up before about the three by two Nuggets worries obviously a little bit of juice because we're dealing with some three-point shooters in that game Blazers grizzlies like they had a plus money. Like I said, there's some three-point shooters on both sides The other one was sacramental bulls. This set minus 132 a little bit more juice than the other two But I prefer taking a little bit of extra plus money for the blazers and the grizzly So it's just a simple promotion For the sports book and then the second part is how do I feel about john the coming over crowded? Or should I spend up on wins a lot? I think john and cominga is an awesome option Both in real like basketball being named to the rising stars game earlier today, I believe And then obviously in fantasy basketball getting playing production as a blade for both states stepping into the starting lineup because dremon green and under equal dollar are both out 5600 consistent production from him for the most part like he's gonna have some of these lower games where He doesn't do as much in the pure scoring And that's something you generally have to embrace which I have to take in a context the offense that he's in right He's not sharing an offense with steve curle He's sharing an offense with clay thompson who may not always be consistent But he is a player that obviously, you know Demands the ball good amount is going to be taking a decent amount of shots And then of course indra wiggins a player that's also going to be taking a good amount of shots So I like cominga for his potential upside especially at his salary Only 50 600 Grimm is asking take the Lakers spread tonight. Is that five right five or five and a half tonight? For the Lakers it's four and a half now. Okay um They're on what a three game losing streak four game losing streak you know four and a half It obviously depends on the on the LeBron news right and and I would I have a tough time betting the Lakers Plus four and a half without the full health of LeBron James because I mean, let's just be realistic about even with LeBron the Lakers are really average this season Even when he's at full health even when he's going for a double double triple double like they're losing games So betting on the Lakers not knowing if LeBron's actually going to play is Personally, that's too much of a risk for me Like I wouldn't go there if he gets ruled in and it stays at four and a half or five or whatever it's going to be I have a little bit of interest in it at that point But I can't I can't be going there right now without LeBron's news because The jazz are back to full strength like Donvin Mitchell is back Rudy Gobiere is back Like if LeBron doesn't play like this is a spot that the jazz could realistically win by 10 points Like I don't think anyone would be shocked to see the jazz win by 10 plus points If LeBron does not play this game considering how average the Lakers are overall Joseph is asking a season long fantasy question. Would you trade Bain and Kelden for Donvin Mitchell just full disclosure? I don't I don't play season long mba. I only play DFS. It's only betting so Desmond Bain and Kelden Johnson for Donvin Mitchell I mean, that's a tough question. Like I don't I don't know your format I don't know the scoring all those things but I like the I like Kelden Johnson I like his ability to just generally fill up the stat sheet on a nightly basis And I would assume that's like pretty pretty Like valuable overall and Desmond Bain with the three-point ability that he has is always good Donvin Mitchell is great But you know two consistent starters ahead of one is something I would probably side with I would I would keep Bain And Kelden Johnson Again take that with a grain of salt. I do not play season long basketball. I haven't in Probably a decade at this point. So it's not really my thing Uh, so tonight's slate again LeBron James news circling back on the Lakers spread question That's something we're waiting on because if he doesn't play again, you want to swap to Anthony Davis who's only $200 more That's where you should be going Uh, none of that's important So you want to be you know focusing in on the LeBron injury news if you're considering playing him You want to be focusing in on a tight group as always a tight group of value plays A consistent group of you know top tier studio and paying up for I am siding personally with Murray Going to de rosin and vooch as my favorite three, you know trying to get to yokich as well You Really have to find some value if you're trying to roster these three stars three players above 10,500 is tough to get to but It can be done. You know if we're gonna be getting Grant Williams in the starting lineup again 3600 Uh, we also potentially get jade and mcganiel's in the starting lineup Going to okc for erin wiggins at 3600 I mean, this is a usable lineup 5400 remaining. You could you know find some mid-range plays that you like You know wouldn't wouldn't be the worst call in the world to go to someone like set curry newly acquired for the nets You know 5300 per player remaining and you have Murray de rosin and yokich loaded with value options This is gonna be a fun slate. Like if you enjoy If you enjoy like building lineups and and you know figure out roster construction Like who's going to be popular on a nightly basis like this is a fun fun slate DJ's asking should I use bones highland as my sixth man? So this is always a fun question who we're going to be seeing as like a player that comes off the bench That has some scoring upside bones. Highland. I think it makes a case tonight Now monti morris. He is ruled in if I remember correctly for He's available to play so monti morris Is available to play for denver. So bones highland. I do think is always a viable option Coming off the bench for the nuggets He takes a good amount of shots a good amount of them are coming from downtown and that Scoring upside and his ability to exceed value quickly will always be there tht could be in the mix Tht could also potentially be in the starting lineup for the lakers if we do see lebron ruled out So initially he would not be the option. I would be going to Right from the jump for this question Lonnie walker always a player can be going to cam thomas We have seen a salary jump up a little bit So it may not be like specifically what you're looking for if you're looking for a little bit of a salary relief type play You could be going there Malik beasley always an option, especially if we're going to be seeing Anthony Edwards ruled out tonight You know a little bit of a shorter bench Because we don't see a whole lot of these players at all play at all for minnesota So i'd be going to bones highland perfect example cam thomas potentially for the nets tht Maybe you know, I would like to see some lebron injury news before deciding on that which is obviously tough On cam johnson always one of my favorite players to go to for the phoenix suns Good amount of three point attempts per game filling up stat sheet just a little bit Scoring boost for them off the bench is always a good option and realistically if If cam johnson saw 30 plus minutes because they rest paul and booker because they're up by so much It's a 15 point spread right 15. It's up to 17 points now It was 15 points 50 it opened at 15 points If the suns blow the rockets out and they rest paul and booker You know the few days before the all star break I don't think that's too much of a stretch and cam johnson sees 25 30 minutes off the bench Just because it's such a blowout. He would be a player. I would be Very excited to have in my player allocation at only 44 How are you on xyre williams as a salary saver? You know, he's okay in the grand scheme of things He's a he's a player that really doesn't always move the needle for me memphis xyre williams Obviously the salary is great The problem is he's not a shooter really for memphis even when Jamarant is out like he's not doing a whole lot. He's well under one fan two point room and even in ideal scenarios Like I guess this game against new york. He was at one fan two point room But very very thin production from him even when jaw is out like we know the ball is going to go into death Mbane, we know it's going to go into jaren jackson So I am not on xyre williams like him eric gordon You're like tony snell at times if you've been playing nba dfs over the past few seasons gordon snell some of these players Just not players. I traditionally go to they just aren't primary scores You we need like a like a top 1 percent outcome for them to like really have an amazing game to truly be worth it Like pj tucker some of these players. I just like don't enjoy going to and More often than not I make it a case to Actually remove them from my player pool It's not just like I let them sit there in the optimizer and let them magically appear in a number of lineups like I actively remove them from my from my lineups trend in wadford He's okay again another one of these players that You know you can be getting some production out of you'd be hoping for the game environment to like kind of run Its course in order for him to reach value But more often than not he's gonna be under 20 fan two points So if you're running a lot of lineups and you want to mix in some of these players, that's fine You want to add them to like your core of value plays that's fine, but if you're running like one Three you're doing like one lineup you're doing three max five ten like these players probably don't make the cut for me xyre williams wofford gordon You know whoever might be like these players are very very fringed to begin with and Troy brown He's a player that's playing a decent amount of minutes for the bulls You know up in in the low 20s mid 20s in some of these games. He's still barely producing. So Not players. I have a whole lot of interest in on most nights So again LeBron news very big on tonight's slates, uh, then glee that news is actually, you know pretty big for Uh, the toronto raptors. He's still listed as questionable. Uh, if he's out you want to be focusing in on Gary Trent's more possible siacum John Moran didn't play last night mean update on him We'll only go back to jasmine bane anthony edwards If he's out we should be seeing jade mcdaniel's get the start And lebron james if he is out we want to be going to anthony davis very simple swap again between davis Going through davis from lebron if you're playing them at the power forward position So keep that in mind keep them at power forward. So we get this uh injury news since it's on the west coast As a recap so far for the betting I took the over on anthony simons over 30 and a half Points rebounds assist combined over on simons. I took the bulls minus six And the over on kuzma points rebounds assist which is sitting at 36 and a half. So those are my three bets so far for tonight Next week the mba is still on break. So we will not be here We will pick up following the all-star break as always Wednesdays except for next week on the fandal youtube twitch twitter and facebook page You can follow me on twitter at dfs underscore tom till next time. Good luck in your contests


My name is Steve Cervantes. I don't live around here anymore, but I find that Emerson, used to play Emerson. I'm from Rhodes Junior High School over in the west side. So my daughter's involved with the schools, so she's out there. All here she comes down there. She's passing out flyers or whatever you do. Anyway, I got a story about the whole breach. Oh, besides, I used to work right here on Sam's School. I don't know if you're familiar with Sanatana Machine and Supply. Right after I graduated from high school, my major was machinist. And I've been a machinist all my life. And that's where I started at Sam's School. The building is still there. When I come downtown for the events, I see the whole building because there's a parking lot there. Anyway, that's an old building. I like all these old buildings and stuff like history.  In fact, when I passed by any street around here, I see old buildings. Can you imagine who lived there or what happened? Anyway, there was this town somewhere in the country, and they had a steel bridge like that. So they started tearing it down, and the whole community, the whole people around the town came on. Hey, what are they doing to the bridge? Oh, they're going to tear it down. They're going to build a new one. So I said, so the people said, wow, that's good, but we can get the money. No, don't worry about it. Somebody funded the money to build the new bridge. It's an old steel bridge. We're going to tear it down. So sure enough, they start tearing it down. People come by, look, they're doing a good job. They're going to start building a new bridge. So they raised the whole bridge.  Anyway, one month passed, and then two months passed. Hey, what are they going to come back and build a new bridge? They stole the bridge. No. Okay, here, they had a chance to do the same thing. They restored it, right? So God's saying, hey, just keep the bridge here because it's still here, right? So that's my story. Okay?


Lisa Cameron is a UK politician or member of Parliament. She regularly attends Westminster where she makes the case for her constituents across Scotland. She's also important to the world of crypto because she's the chairperson of the Crypto and Digital Assets All-Party Parliamentary Group. In brief, this group is responsible for guiding the UK on its mission of becoming a global cryptocurrency hub. So I took a break from moderating and comparing at the UK's major Bitcoin conference called the Bitcoin Collective to interview Lisa. She then made the case for Bitcoin and crypto in Parliament just days later. She requested to meet with Jeremy Hunt, the Chancellor of the Exchequer. He's arguably the second most important person in Britain after the new crypto-friendly Prime Minister, Rishi Sunak. Firstly, I mean, we just came off a talk here with Samsung now. We're talking about how governments can run with Bitcoin and cryptocurrencies. But I wanted to ask you just a general thing. Why are you here at a Bitcoin conference? Well, it's in Scotland, which is my home turf. So that's a big draw for me. As part of my education as chair of the All-Party Parliamentary Group, I'm engaging with the sector and the industry in the widest way. So I have spoken to companies who are involved in CBDC, stablecoins. We've looked at crypto tokens and Bitcoin is obviously part of the sector. It's very, very important for me to understand the differentiations, but also to understand, you know, how the technological advances and what's happening internationally as well as, you know, the potential for the UK can be taken forward. Interesting. And you mentioned lots of these cryptocurrency buzzwords that we all deal with now on a daily basis. What for you personally is the difference, say, between Bitcoin and crypto, or Bitcoin and NFTs? Or is there a difference? Or do you see them all in the same sort of light? Well, as part of the chair of the All-Party Parliamentary Group, we're looking at the sector as a whole. So we're not sort of saying, well, we're only going to deal with Bitcoin or we're only going to deal with... Because consumers, who are our constituents, are engaged with the industry right across the board. And our key sort of issue is about consumer protection, is about looking at regulatory frameworks moving ahead in the UK, which in my understanding and from the session we've had to date in the Bitcoin conference, you know, some of that relates to Bitcoin, some perhaps not so greatly because of the decentralized nature of it. So what we need to do, I think, is firstly in the parliament, have an understanding of all of these things, which is no mean feat, you know. It's quite a different language, as I said. You know, when we first had the discussions in the parliament, money was being grouped into this sector and Fiat, you know, like we were explaining to the MPs that Fiat wasn't a car. So, yeah. Really, they're looking at Fiat 500s instead of Fiat money. It's a different terminology. We are, you know, having to think of all the acronyms that are used and incorporate them now into our understanding and knowledge. So we, and I include myself in that, we are on a learning curve. And it's just very, very important because the UK government has a policy vision that the UK will become an international hub of quick currency and digital assets. So we need to understand the workings of that, the challenges ahead. We need to understand how to put consumer protection at the core of that as well, and what businesses need in terms of regulatory clarity. And I need to understand how Bitcoin fits into all of that, or how it doesn't, how it might be separate. Okay. Yeah. Yeah. And that's amazing. It's interesting to see the level of understanding. Like, have there been any conversations that have been quite striking, you know, but brought up Fiat? I recently had a conversation with an energy company, and they were asking where you find the Bitcoin when you mine it, because they thought it was a physical, you know, mining operation. What sort of level of understanding is there currently in Westminster? It's growing now. The group's been running for about seven months now. We have our inquiry ongoing. What we find is that, you know, when the group was formed, obviously we get a lot of interest from members of the Parliament, members of the House of Lords who are already aware of the sector, who are also, you know, perhaps financial, you know, have financial backgrounds in perhaps their formative careers. But now what we're having to do and want to do is scoop up the educational evidence base and understanding of those MPs who perhaps haven't been aware at all much of the sector, and who maybe have formed their basis of understanding solely on sensationalism, rather than an evidence-based information-based, you know, sort of understanding on knowledge, because when we come to have debates in the Parliament, we want those to be very balanced and evidence-based moving ahead. Yeah, which is the hardest part, right? Cutting through all that noise and that sensation, isn't it, that you bring up? What about, personally, can I ask, have you ever, say, bought an FT? Have you ever bought Bitcoin? People have asked me that, and no, I don't have... As chair of the group, I feel it's important that I have a kind of objectivity and distance from that, in terms of, you know, my own personal... But you have fiat money, right? You don't have a fiat car, but you have fiat money. I don't have a fiat car, no. I do have fiat money. Yeah, and that's a very good point, but just in terms of making sure that, you know, anybody who's looking at the work we're doing and scrutinising it, which, you know, perfectly the way it should be in terms of democratic politics, is not thinking that I have some, you know, personal investment in this that's driving it ahead. We're looking at it very objectively, and, you know, we want to hear from all parts of the sector. We want to, you know, have an informed approach going ahead, and we don't have any preconceived emotions of that. What I want to do is learn about the technology and learn how it's used. But I think, if I, all of a sudden, became, you know, Bitcoin maxi. Yeah, exactly. Well, there's a new part of technological language that I'm learning, Bitcoin maxi. Yeah, Maximalist, yeah. It means you only really care about Bitcoin and everything else you see, et cetera. Right, okay. It's what Samson was talking about. We're going Pierre Poirier of the Canadian Minister. So, if that were to happen, then I think it would skew the report. It could, you know, mean that I'm less objective.  You are technically. To buy the wine that was talked about in the session, yeah. Wow, you know, I think we all know, particularly from the pandemic, that finance is shifting. Most people pay for things using digital technology now and actually, you know, fiat notes, pints, currency. Yeah, that's all shifting and progressing. And Web 3 and the innovations moving forward and the whole industry of digital assets is transforming. And we have to really understand that in the parliament and we have to make sure that we support the vision that the UK is a hub of the future. Because otherwise, if we don't, then we'll fall behind. Yeah. And I think so there is tremendous potential for innovation and growth and jobs of the future. And we see that already developing and fantastic research base here in terms of technological expertise, universities right across the UK through the helm of innovation and technology. We want to really, you know, maximise all of that and be a place that people want to do business for the sector. And I think, you know, part of my role is, it's almost apolitical in a sense because we have MPs and members of the household of all parties is about just making sure we have the education piece. We understand what's needed in terms of clarity moving ahead. We can keep prioritising these matters in the parliament because they weren't spoken about before. We had a first debate just a couple of months ago. The first debate on these matters. Yeah, yeah. So it's so early. Yeah. Okay. So, and I raised a question on Monday to Jeremy Hunt, the new Chancellor, mentioned about the conferences that are ongoing in the UK this week. How important they are. And we're going- Okay, he's good about these conferences. Yes, I did. I mentioned the, yes, I mentioned the Digital Assets Summit and also the Bitcoin Collective Summit. Yeah. So I mentioned both and they're speaking of both and he's aware of it. We're meeting with the Minister to take forward his understanding and engagement with the sector and, yeah, Westminster should be open to this technological discovery advancement that wants to be at the helm of it. Very good. I think it's lovely to finish on. Thank you so much, Dr Lisa Cannon. Thank you. This has been a Cointelegraph interview.


Welcome to our first time chat and thank you very much. I have nice plants. I have very nice plants and they're giving food and stuff like this. So thank you for noticing. I like my plants. I like my space here or our space here. Thank you very God, all the God. Dain, what do I grow? I grow. Dain, thank you very much for the follow. Salute, salute. What do I grow? What do I grow? Just in line of sight. I'm gonna go from here this way. We got lettuce. We got sage. We got raspberry. We got gooseberry. We got snap peas. I got grapes. I got green onions, strawberries. I forget the name of these flowers. They're really good. I forget the name of the tree. Tomatoes, lettuce, chard, kale, shiso. More flowers, shiso. I got grass for the kitty cats. This plant over here that you see here, let me pull it out. I'm gonna show it to you. Ready? Ready? Four cannabis plants growing and we've got a whole bunch of stuff back there. Here I'll show you my snack sometimes. Well, every day I come out now snack on We got black currants. We got cucumber. We got zucchini.  We got these flowers. I forget what they drain. No, not just Cheryl knows this. These guys are really good. They're spicy. You can eat them. You can eat the leaf too. The snap peas. Really good snap peas. This one. Nice and flat. That's why you use this one, it will become this one. What you like this is really good too. I've become a professional at eating these.  Really nice. Delicious sugar snap peas. We lost one. We lost a little guy in there. So it's nice. And I'm growing tobacco too. David, thank you very much for the follow. Salut, salut.


Hello it's Missy thank you for watching! For today's video I wanted to share some high bun type hairstyles. I feel like I haven't done a video like this one in so long so I hope you enjoy it. Let me know in the comment section which of the styles is your favorite and of course if you're new to my channel be sure to like and subscribe, click that notification bell and then you'll be notified every time I post a new video and you'll never miss one and let's get started! For the first hairstyle I'm going to brush all of my hair up to the crown of my head. I want to place all of the hair where I plan on positioning my bun so I'm going to smooth down the sides and then use a hair band to tie all of the hair together. Then once the ponytail is tied off I'm going to take a small section of hair and wrap this around the hair band. I want to make sure the hair band stays hidden and just in case the bun doesn't cover it I'm going to wrap a section of hair around it and then just secure it underneath the ponytail using a bobby pin. Now I'm going to take the ponytail and divide it into two equal sections. I'm going to create a rope braid with the hair so I'm crossing the left strand over the right strand while simultaneously twisting the two sections in the opposite direction towards the left side. So I'm twisting the two sections while crossing the left strand over the right strand and I'm going to continue that pattern until the braid reaches the ends of the hair. Then once the braid reaches the bottom or as close as I can get without it becoming too tedious I'm going to take a clear elastic band and tie off the end of the braid so it doesn't unravel. Now using this braid I'm going to create the bun so I'm going to take the hair and wrap it once around the hair band clockwise. I want to make sure the bun is going to be easy to pin so I'm laying the braid against my head as much as I can so it's more flat instead of poking out from my head and now I'm going to pin down the braid so I'm using bobby pins and I'm just going to slide this into the hair between the braid and my head wherever I'm holding down the hair and I like to make sure they're sliding inwards towards the hair band so they don't show or stick out at all. Then once the hair feels secured in place I'm going to take the end of the braid and pull on the edges so the twist becomes really big and puffy. I need this to be more full to add volume to the bun so I'm carefully pulling small sections of hair up and slightly out of the twist until I feel like it's big enough to use for the bun and then I'm going to lay this twist against the right side of the bun and tuck the very ends underneath. Then using bobby pins I'm going to slide these into the bun so they're gripping a bit of hair from the twist as well as a little bit of hair from my head so that will hold the twist in place and once it feels like it's pinned and won't fall loose I'm going to continue pulling on small sections of the twist just until the bun looks more symmetrical and I like the way that it looks. For the next hairstyle I'm going to begin by dividing off a section of hair starting from right in front of both ears and then going up to the top of my head. I'm going to take a clear elastic band and tie this section off and then I'm going to clip this section out of the way so I can use it later on and now I'm going to divide off a similar section from right behind the first one so starting on my ears I'm going to create a diagonal part line up towards the top of my head then I'm using another clear elastic band to tie off this section of hair as well and now I'm going to let down the first ponytail that I created and I'm going to divide this hair into two equal sections. I'm going to take the second ponytail and bring it up between the two sections of hair and clip it out of the way then I'm going to take the two sections from the first ponytail and combine them back together with the remaining hair that's left down on my head. I'm going to use a clear elastic band and tie all of the hair together right underneath the second ponytail and now I'm going to take the second ponytail and divide this hair into two equal sections and then I'm going to bring the third ponytail up through the middle in between the two sections of hair and clip this out of the way and now I'm going to combine the two sections back together and tie this off using another clear elastic band and then I'm going to let down the ponytail that was clipped away and at this point I decided to go back over the first looped section and pull on the edges so I could see how big and full I could get it so I'm just pulling small sections out of the elastic just slightly so it's two to three times bigger and the more you pull the bigger your bun will be and the less you pull the smaller and tighter the bun will be so now I'm going to take the top ponytail and divide it into two equal sections and I'm going to wrap these around the bottom ponytail and then tie them back together underneath with a clear elastic band and then I'm going to go back over the two new looped sections and pull out the edges so they're really big and as puffy as the first section and now I'm going to create one more looped section of the braid so I'm dividing the top ponytail into two sections wrapping them around the bottom ponytail and tying them back together using a clear elastic band and I'm going to pull on the edges of the looped sections so they're the same size as the other loops and now I'm going to take the ends and tuck them underneath the hair to form the bun along the top portion of my head and then I'm going to grab some bobby pins and pin the ends underneath so they stay in place I couldn't decide if I wanted some of the ends to stick out from the bun just for a more casual style but then just decided to keep it a little bit cleaner so I tucked everything underneath the looped sections and just pin them in place with bobby pins for the third hairstyle I'm going to prep the style by pulling out sections of hair to frame my face so I've placed my part near the center and I'm dividing out hair from along the hairline on both sides of the part then keeping my part in place as much as I can I'm going to brush the rest of my hair together placing it high on my head near the crown I'm going to do one of my favorite style buns and this is similar to styles that are floating around tiktok but I've done this so many times I've kind of figured out an easy way to hold my hand so I can do it really quickly so what I like to do is hold the hair in my right hand with the hair band around my right hand as well and then using my left hand I'm going to pull the hair band part way over the hair so what I like to do is grab my ponytail with my pointer finger and thumb and I'm going to hold the hair with these two fingers and then essentially push the hair towards the front of my head and you can move it without using your hand like this but I just think this is the easiest the more I did it the more I just kind of developed this way of holding the hair but I'm just going to twist my left hand underneath the hair back towards the back of my head and this will bring the ponytail forwards and then I'm just going to open the hair band using my left thumb so I can wrap it over the hair again so then I'm going to use my right hand to pull the band completely across the bun for the second wrap then I'm going to pull on the hair to shape the bun so it's bigger more full a little more symmetrically placed on my head and with the end sticking out I'm going to loop these back towards the middle of the bun and tuck them into the hair band this will help close off any gaps you might have in the bun and you can also use a couple bobby pins to help close off any spacings between the hair and pin up any ends that are left poking out thank you so much for watching I hope you enjoyed today's video let me know in the comment section which of the styles is your favorite and be sure to like and subscribe click that notification bell then you'll be notified every time I post a video and you'll never miss one and I will see you next time


Watch he work Let me tell you this here about this Madden's video games are video games. You know saying it's video games, bro Have to have tea is another dude.  He's never beat me. No, oh my god What's up with kids and tight just know how to win all the time Come on you throw me the football already, man. Yo, don't little give it up. Give it up buck. It's over. It's over This whole trash Bro, it's just no way right now these fourth downs that cuz you got it, bro Let's take just find your three somehow find it somehow Thank you, man. Oh, how do again. Come on. We're just the know how to Superstars, huh? I can't get off the field here boys. Let's get off the field like men here, bro Save my life chat save it. That's what I'd be trying to tell myself that though eventually do a pic, but Possession catch the yards that are out there, dude. I got bagged right there. Ah, not to play ball Come on, man. Love this game, bro. I just love it. I love it, dude Good it's good and call right there, buddy. Good call. I mean Calvin, you're Bro, I shaved you down. Come on, man. Oh, now it's the run show. Huh? You little now. It's the run show. Oh My god, just You're just a man. We just ran for previous play over and over and over I mean, I was afraid to possession catch that good decision if you ask me Raider my god pretty much is weak one right now.  Come on. Somebody get the home, bro. Somebody got to get home right there, bro.  Well, let's take a chance here boys. Oh, yes, sir Yes, sir ski Yes, sir ski man, they cheat him cuz he can't make a read. That's why they cheat him, huh?  That's what happens.  Let's get this up, bro Let's go man. He lost when he ran stretch on 38 exactly Hulk now You might as well run the ball now you might as well ran the ball because you ran the ball for your life You might as well run the ball now kid. You might as well.  That's why he lost right he lost because the game glitcher not cuz I marched down the field and get a touchdown Right with one time out, right?  I mean he could have caught it But he gave a chance He didn't make it late read and he gave the game a chance to and let me tell you something about this game here Madden 20 Madden football. Let me tell you this boys If you give the game a chance to get it'll bend you over and it won't feel bad about it. All right


Welcome to MOOC course on Introduction to Proteogenomics. In the last lecture, Dr. Karl Klauser introduced you to the basics of mass spectrometry based proteomics. Today's lecture will focus on the crucial steps in sample preparation for mass spectrometry based proteomics and also to provide a glimpse of label based quantitative proteomic approaches. Further, the concepts of peptide spectrum match or PSM's and spectrum library matching will be covered. So, let us welcome Dr. Klauser for his second lecture. Extra things and that might be possible to use in reducing your list of the confidently assigned peptides, okay. Some scoring systems are going to be, but we are almost never these days going to collect one MS spectrum at a time. We are going to stick it in a machine and the machine is going to go and go generating mass spectra as long as it can, alright. So, you start out with to make that automated workflow happen, you are going to start out with a source of material which could be tissue, could be cell lines. You are going to extract that into proteins, then we most often will digest the proteins into peptides, the peptides then go into a mass spectrometer and then this automated system does three basic steps, okay. It is going to separate the peptides chromatographically, eluding them over time based on their hydrophobicity and so this in this description here that run time takes about 120 minutes, okay. At some given point in time, a scan is going to happen. This takes about 10 to 100 milliseconds now. The first thing you would do in a cycle is take an MS scan, you measure the masses of all of the peptides that are present and then you will very quickly collect some number of MS-MS spectrum, a common number to do now is 10. So, it will take the 10 biggest things that it was observed in this MS scan and do MS-MS on them, okay. Today this cycle like this can happen in a second, okay, alright and then this huge collection of spectra that are generated automatically then we will get put into a software program and it will try to match up assigned peptide sequences to each of the mass spectra and then you will have some additional software that will try to take the peptides that belong to the same protein and you get out a list of proteins that were observed and all of the peptides that you have observed with those, okay. This is one of the, I want to say most desirable instrument but that it is most desirable only if you can afford it. It is one of the most expensive instruments and it can do a whole lot of things, okay. The CPTAC program is currently generating almost all of its data on this kind of an instrument but we do not use the entire capability of the instrument, okay. So this instrument, a fusion lumos from thermo, you put ions in here, you then have a way of isolating precursor ions here and then you can do MS-MS by three different techniques. You can do something called high energy, higher energy collision dissociation, you can do collision induced dissociation or you can do electron transfer dissociation, okay. And then you measure things, the spectra in the orbit trap. It is also possible to measure them in the ion trap out here at lower resolution. You can go faster with lower resolution if you go out here, okay. In practice in the CPTAC program for generating proteogenomic data, we generate only HCD spectra and we collect mass spectra only in the orbit trap, okay. So we are not taking full advantage of the instrument. The reason we are using those instruments is because this instrument which does only the things that we really want to, did not become available until just earlier in this year and we started the grants that we were doing a year before that, okay. All right, so this I think of as today the workhorse type of instrument that if one was setting up a lab to do proteogenomics in the way that we are going to describe having done it in the CPTAC program, this instrument would be the one that one would get today. Sample preparation, okay. So in proteomics, these are some of the basic considerations that you have to do in designing your experiment, okay. So quite often going to start with either cells, tissue, fluid, fluid might be blood for example and then there is going to be some set of separations that we are going to choose to do, okay. You have the choice of maybe you want to do some fractionation at the protein level. You might want to do some enrichment or depletion at the protein level. If you are working on cells and you care about mitochondria, you might do a preparation that gives you an enrichment of that subcellular fraction that you are interested in, okay. From the standpoint of proteogenomics, we do not do any fractionation at the protein level, okay. The first thing we do is digest in the peptides and then it is all about separation of peptides after that, okay.  Plasma, the most abundant protein in plasma is albumin, right and it is the least interesting protein. But it is the first thing you want to do is get rid of it, okay. So you use a depletion step to get rid of albumin before you go to peptides, right. But for the purpose of doing cancer proteogenomics, we take our tissue, grind it up, go to peptides and then we are going to fractionate peptides. Typically if you are going to do it offline before you go to the instrument, what you want to do is choose a methodology that is going to give you a different kind of separation than the one that is going into the instrument. So two common ways of doing that are either ion exchange or what we most commonly do now which is basic reverse phase, okay. So that means we are running a reverse phase separation but we run it at pH 10, okay. So separation that goes into the instrument goes at pH 3, okay. Another thing that you want to do is enrichment, okay. So if you are after phosphopeptides, you do not have to sequence everything else to get to your phosphopeptides. So you use something to pull them out, we use a mobilized metal affinity chromatography. If you are interested in lysine acetylated peptides, you can isolate them with anti-acetyl lysine antibody, okay. All right, so in choosing what you want to do, you are looking to make a trade-off among these criteria, okay. All right, most proteomics today is done in a way where there is going to be a digestion step to peptides, okay. Trypsin is by far the most common enzyme to use and it gives you convenient lengths of peptides that are generally tend to work well in a mastectrometer. They have the property that they have a basic amino acid at the C-terminus which is going to give you somewhat better fragmentation than if it is not at the C-terminus, okay. Cysteines can be disulfide linked when they are in a protein. If you just reduce them, they are very hard to chemically maintain throughout your process. So what we typically do is reduce the dis, break the disulfide bonds and then alkylate them with some agent like iotacetamide that then makes them readily detectable, okay. All right, so when you are doing enrichment, here you want to think about whether you are doing enrichment or depletion at the protein level here, then there would be a digestion step and then consider fractionation or affinity enrichment. The reason that you would make all of these kinds of strategic decisions is probably with some goal of increasing your depth of coverage. So if you want to start out with a complex sample and you are only interested in these things that are low abundance, there is going to be typically some form of infinity enrichment involved or depletion of more abundant components, all right. All right, so the ones that are the most common post translation modifications that are, that people can work on that are do by large scale, methods, phosphorylation of course is the most significant one. In our lab we also do a lot of ubiquitination work. This happens, this is done by having a glycine-glycine which is the, starts out on the ubiquitin. So the ubiquitin is covalently bonded to a lysine in a protein. When you treat it with trypsin, it cuts off the ubiquitin but leaves two glycines that were the C-terminus of the ubiquitin, okay. Satellated lysines are something else that we also now are doing routinely in the CPTAC program, okay. So and you can do these by using an anti-stial lysine to anybody, all right. So we also have done this in a way where we don't have to split up the sample and lose, require more sample and then dedicate only some of it to one modification, only some of it to another, some of it to a third. Instead, we do the enrichments one after the other, okay. So the supernatant of what comes through a IMAT column can then be used to, so the things that don't bind to the IMAT column come through. You can then do a next step of enrichment for something else and in our case we've published work on doing those three things in cereal, okay. So you start with less total sample and achieve each of those items, all right. Okay, quantitation and multiplexing. The almost anything that we do in our lab today in proteomics and quite a lot of labs are trying to do things that are quantitative, okay. And the basis of doing something quantitative and having some statistical power requires that you have replicates, okay. So not only do you want to have replicates but you typically want to compare two conditions, at least two conditions. In examples of this might include wild type versus mutant expression, treatment with a drug or without a drug or capturing something with a bait or not. And then most of what you detect is probably going to be unchanged between the conditions and you're looking to do statistics to recognize some subset of things which change between the conditions that you do, okay, all right. What sort of experimental design considerations that you put into this, okay. I'm going to show you three different techniques. One here is called label-free where you basically combine the samples at the end, okay. And then I'm going to show you two labeled techniques. One is silak, stable isotope labeling of amino acids in cell culture. And then the third one is something called a TMT or ITRAC where you're using a chemical labeling agent. You then are going to combine the samples and then put them into mass spectrometer. You do MSMS, the quantitation comes at the level of the MSMS spectrum, okay. And this technique here you combine earlier in the process and the quantitation comes at the MS level, okay. Multiplexing-wise you can do three things, three samples at a time pictured are only two, okay. You can do light and heavy. The third one would be medium, okay, all right. The TMT-10 reagent you can put together 10 samples. If you have an ITRAC reagent, there's actually two ITRAC reagents. One is called ITRAC-4 and the other one is called ITRAC-8, okay. So that tells you how many samples you can put together. There's also something called TMT-6, okay. And I will get into some of the differences in what you have to have to be able to do those kinds of experiments, okay. So here are some of the features about this. There's a lot more time to do an experiment this way, a lot more instrument time. Here there is some loss of accuracy in the quantitation due to compression that I'll talk to you about. The reagents can be expensive, okay, all right. Here you have less potential to do flexing and in order to get a heavy label, you have to be able to add that to the cell culture that is going on. So that means you can't label humans, okay. So you can, this is really suited to working with cells in cell culture, okay. All right. And the quality of the quantitation is shown here and would be highest over here, okay. Why is it highest over there, okay. Ideally when you're going to mix things together, you'd like to mix them as early in the process as possible so that any of the experimental variable, variability that happens, happens to all the samples together, okay. But because of the way you do the experiment, you can't necessarily mix things until a later stage, okay. So in the case of chemical labeling, you have to mix after you have done digestion. But if you do it in cell culture, you get to add, do the combination way back when they, just after the cells have grown, okay, right. And so I think I've already said some of the pros and cons about this. So let's move on, all right. Let's go straight to what happens when you do chemical labeling approach, okay. So the idea here is this, you might, here I'm illustrating TMT-6, okay. So you would have six samples. You lice each of those, the samples, that gives you proteins. Then you reduce and alkylate, trypsin, digest the peptides. After you have peptides, you use the TMT labeling agent. These are amine chemistry-based reagents, so they're going to put a label on the side chain of lysine and on the end terminus of the peptide, okay. And so the reagent normally comes in six colors, okay. These are actually masses, and the masses shown here are the reporter ion masses that are present in the MS-MS spectrum, okay. So then after you do the labeling, you mix the samples, and then you have six different things labeled. The purpose of doing it this way is the labeling reagent causes all of the samples to have the same mass, okay. And the label is going to have a different mass, but only after you do MS-MS, okay. So the signal that you see in an MS-1 scan is the sum of all of the six samples, okay. Which is good, right? It means you get more signal when you combine the samples, okay. And then after you fragment, you're going to have reporter ions that allow you to get the peak height that is shown here is going to enable you to do the quantitation back to the samples that they came from. All right, this is the chemical structure of the label, right. And shown where the asterisks are is where you would put C13 or N15, okay. And in order to do the labeling, you're going to put in five labels. But depending upon where you put them, you can end up with a 126 ion. If you put, I have another slide that will show you where. But the idea is you're going to put them in different places so that you have different labeling capability, okay. Now, because I told you at the very beginning, you could tell the difference between N15 and C13, right. If you have high enough resolution, you can separate and you can get 10 different things, okay. And that is all going to go back to whether there was a label on this nitrogen or C13 in this position, okay. So now this slide is harder to see. But the dots show you where the labels are for each of the different reagents, okay. All right, and then the things here colored in black correspond to the reagents that are for TMT6. The additional things in red are the extra channels that you use for TMT10, okay. Unfortunately, it's a little bit complicated, okay. So you have some impurities that you have to deal with in this thing. And there are two types of impurities, okay. One sort of an impurity comes from how pure is the C13 that you start to put in the label, okay. You can get over 99% pure C13 to incorporate these days. But there's some level of impurity. Same is true of nitrogen 15, okay. But there's a second set of impurity which is, this is in the unlabeled positions, okay. So this is over here. There's these carbons over here that are naturally occurring levels of C13, okay. And so if you end up with a C13 in one of these positions, it's going to be one carbon higher in mass than it would be, okay. So when you obtain the reagents, they also give you a set of correction factors, okay. That software will apply to correct the intensities to account for the impurities present in the label, okay. If you obtain data from some public repository and you want to reprocess it all from scratch, make sure you get the correction factors that are provided by the people who generated the data. Unfortunately, they don't always remember to give you the correction factors when they deposit the data somewhere and you might have to send email asking for them and hopefully someone can write back and give them to you, okay. One of the things that we try to do from our lab is always provide these, but I often have to chase down the people who did the experiment and say you need to provide these before we can put the data in a public repository, okay. Those correction factors are then used with an algorithm to apply them and correct the intensities. This is a publication that's about ten years old. This is, we use the same method that they describe. We don't use the exact same software because this publication is old enough that it only applied to iTrack4. We've modified it to be able to work with TMT6 and TMT10, okay. All right, there's another sort of set part of complication in working with TMT quantitation. And that comes down to interference, which goes back to if you fragment more than one thing at a time, okay. And so what I've tried to do is draw a cartoon here to illustrate how this works, okay. If you had two peptides of very similar precursor mass that were present at the same time, and let's say one of them is uninteresting. There's no one of the six samples that has either up or down regulated levels of protein. But in the sample that's right next to it, it has up regulated levels of the red, okay. So there's way more red in this one than there is in the other one. If you're using an older instrument, you might have to set the precursor mass window to be two Dalton's wide, which would cause both of these things to be transmitted at the same time, okay. The labels produce the same reporter ion masses, and you can't tell which one they came from, all right. So if you were to transmit this whole thing, then you would have a reporter ion set that looked like this only when the data came off the instrument, it wouldn't have this white line through it that allowed you to tell which one it was. You would just have the sum of these things, okay. If you were able to use an instrument that had a narrow precursor window, then what information you would get would be just derived from this one peptide, okay. All right, so if you put the quantitation together and you combine these things, the ratio that you would calculate, if you calculated the red divided by the pink, I'm sorry, let's call that orange, you would get a ratio of 2.5. If you had only the one together, you would get a ratio of four, okay. So the ratio of four is what you wanted to observe, but it is compressed to 2.5 because of this effect, okay. All right, so this is just an example of what might happen, okay. And so that there's a couple of things you can do to deal with this, right. The first is you could do a better experiment, right. If you have an instrument that allows you to do a better transmission, this all of the CPTAC work that is gonna be presented later in the week and is already published is all iTrack data run on a Q-exactive instruments that at the time had a window of two developments for precursor transmission. What you can now routinely do on a Lumos instrument or a Q-exactive HFX is run 0.7 MZ tolerance or window width. And so you would be able to in this kind of case transmit only the one thing, okay. The second thing you could do is you could have data analysis that would go back and look at all your MS scans and say, ah, if we've got this thing, let's throw away that data point, okay. And because we're expecting most of our proteins to be detected by multiple peptides, we have some way of taking and recognizing that some data points are better than others and so we can exclude those, okay. So a common thing to do, different people do this, but they don't all call it the same thing, which is to take some measure of whether how many things are here and what's the relative abundance of those things that are there and when the relative abundance of those things is high, then you throw away the data point, okay. Now that's an approximation because although I have shown you in this cartoon example that the ratios of the MS1 peaks is the same as the relative ratios of the reporter ions, that's not always what actually happens, okay. When an individual peptide fragments, you're gonna get some reporter ion signal and some sequence ion signal. But sometimes the balance is like this, sometimes it's like that, okay. And so even if this peak right here in the MS1 scan is taller, it doesn't necessarily mean it's gonna contribute more reporter ion signal, okay. All right, so that's some of the uncertainty that's present in this type of data and getting better at this is there's room for improving our data analysis, okay. All right, all right. Scoring peptide spectrum matches, all right. All right, so this slide I already showed you once. It was several equipment failures earlier. All right, but the idea is that you're gonna take a sequence database and your experimental spectrum. You have programs that are gonna approximate what the spectrum is expected to look like and then score them. These are some examples of some names of programs that do this, okay. If you are gonna design an algorithm to do this, these are the kinds of things that you would have to think about, okay. And when you start to just look at other programs, these are some of the things that you could think about in terms of evaluating or reading about what they do, right. So, but they're all gonna one way or another have to deal with these kinds of things, okay. So there is going to have to be some step. Maybe it's not within the search program itself. Might be a program that you can run ahead of time that will do peak detection, okay. And it's gonna do these kinds of things. It's gonna do deisotoping. It could assign fragment charge and do some sort of signal to noise processing so that you're hopefully trying to only use a signal peaks, all right. You have to have, when you design the algorithm, know what fragment ion types are possible, okay. And when you start to use a program, you often have to choose what instrument type it is that you use to generate that spectrum. And when you've done that, it's gonna behind the scenes be consulting a configuration file that's got appropriate things like what ion types are possible for that instrument and potentially some different scoring values for the different ion types, okay. All right, then, when your algorithm also, not only does it have mass information, it has intensity information. Today, search programs generally make not very much use of intensity other than to say present or not present, okay. With some of the machine learning approaches that are starting to be imposed, one of the goals of those is to make better use of intensity information, okay. All right, you're gonna have to choose some fragment tolerance units, okay. I told you the resolution was different across the mass range in certain instruments that is particularly true in orbit traps and time of flight instruments. The mass accuracy is also different across the measure, across the mass range. And so we use different units. If you use parts per million unit, a typical value of a good mass accuracy on a high-res instrument ought to be plus or minus five parts per million, okay. And you would say that across the entire mass range. But when you convert that parts per million into Dalton's, that means it's a wider mass, I'm sorry, wider mass window at high mass and a narrower mass window in Dalton's at low mass, okay. So if your instrument data has your mass accuracy specifically in units of PPM, ideally you would like to use a search program that could also support mass accuracy in PPM units, okay. But it is actually quite common to use a program where it only has Dalton mass accuracy. And so what you have to do is compromise and set the tolerance to only use the high mass one when you should be able to in principle use it at lower mass and have a narrower tolerance, okay. All right, most search engines produce a score that is the primary score that's used to make most decisions, but along the way they might calculate extra things and that might be possible to use in reducing your list to the confidently assigned peptides. Okay, some scoring systems are gonna be dependent upon the size of the database. Others are gonna be only dependent upon the scoring of the ions and a particular sequence. And if you take that sequence and put it in a big database or a little debated database, the score is gonna be the same, okay. Some search engines will, however, take the size of the database into account. All right, so that's what you have to do if you're designing an algorithm, you consider all those things. If you're gonna use one, you have to consider these kinds of things, okay. You have to choose a database, okay. Most of the time today, there's also an opportunity to somehow do a decoy database that is used to calculate false discovery rate, okay. As you read literature, you will find that there are certain groups that always allow for partial enzyme specificity, okay. While other people may require that fully specific. So the trypsin had to cleave on both ends of the peptide. If you're using a partial enzyme specificity, that increases the search space that the spectra are gonna be matched against. The program is gonna run slower and you usually have to have higher score thresholds to meet your FDR, okay. When you're gonna choose fixed and variable modifications, you wanna choose things that you can expect to find in your sample. And if you are interested in these things that are rare, especially if you choose many of them, it's gonna slow down the search. And I have a slide that a little bit, we'll talk about expansion of search space, okay. Then you have to choose, like I said, precursor ion tolerance and fragment ion tolerance, okay. All right, this is how the spectrum is scored in my software called Spectromil, right. And this up here is shown with the, all of the peaks that are present in the spectrum as it's generated from the instrument. Instrument doesn't have it colored blue, red and green though, it's all black, okay. All right, there's a preprocessing step that does peak detection that does these three, these several things, deisotoping, signal noise thresholding, removes apparent reliance, neutral peaks. So these are the only peaks that are left, that are subject to the scoring, okay. The scoring has three components to it. There is a positive component that means the mass matches a fragment ion type. That match, the score of that is independent of the intensity, but it is weighted by what ion type it is, okay. There is a bonus for having composition information like ammonium ions. And then there is a negative portion of the score that is for peaks that are not assigned, okay. And so basically a tall peak in a spectrum that's unassigned, that's bad, right. That suggests that you have an incorrect interpretation or you've got multiple things that are being fragmented at the same time, okay. The different ion types have different scores, B and Y have the highest score, they have score of one.  All right, so you do all of those things and you end up with a score. In this particular case the score is 12. The peak detection will produce no more than 25 peaks. The maximum score is 25, okay. All right, now something that's quite a bit more different and less intuitive is something like one of these scores that is used a probability-based approach and this is the binomial probability equation. It is the basis for scoring in the Andromeda search engine that's part of MaxQuant. Roughly the same approach is used in mascot. And the way this works is that all ion types are given the same weight, okay. And in order to calculate the probability you have to account for the chance of there being a randomly matched peak, okay. And the way that this is put into the binomial probability essentially comes down to breaking up the mass range into 100, I mean 100 Dalton chunks and you say if we're going to look for say six peaks then the chance of randomly matching would be six out of 100, okay. It may not be immediately obvious, but that also suggests that the mass tolerance you are allowing was plus or minus a half a Dalton, okay. All right, now in practice MaxQuant has allowed you to specify a fragment ion tolerance, okay. But that is not used as part of the scoring, okay. And up until about one or two years ago mascot did not allow you to use part per million as a fragment tolerance, you had to use Dalton's, okay. And it's because of the way that the scoring is built into the probability, okay. So from my point of view, the probability is not true probability but the scores are still effective, okay. And the reason that the probability is not true are for the reasons that I've listed here and I've already talked through, okay. All right, now what I wanna do to show you here is a contrast in this is what you would do if you knew what that peptide fragmentation was gonna look like, okay.  The particular case that I'm showing actually is one of these things where somebody is trying to demonstrate that the thing that they observed in a complicated experiment, they made the synthetic peptide, the spectrum looks almost the same, you can calculate a spectral similarity metric and it passes the threshold and they can say see, this is what we said it was, okay. All right, so the equation that gets used here is a dot product score, there are a few different variations on this and I'm not gonna go through the math but the point is that you're really taking advantage of the intensities and you're not allowing for all possible fragment ion types that could occur to a peptide, you're only allowing for the ones that actually occurred to generate the reference spectrum, okay. So software programs that do this kind of spectral library search are listed right here, okay. The FDR method that's calculated is sort of today not thought of as being as statistically rigorous as what is used for database searching and as I would characterize that as a work in progress to be able to do good false discovery rate calculations, okay. Now it's also the case that in order to do this effectively you have to have a good set of reference spectra to match to, okay. And one of the things we found in our lab is that once we've got just a good reference library somebody came up with a new chemical labeling agent and we switched it over it and they all they fragment all differently and now we have to start over, okay. All right, but after we've done a lot of work somebody can collect all of our spectra and then use that as the basis for creating a library, okay. All right. Now let's talk a little bit about localizing and post-translational modification set. All right, so what I've got here is a MSMS spectrum of a phospho peptide. This is not two spectra, this is one spectrum. It's just labeled two different ways, okay. The same, you can see it's the same peptide sequence. Hold on. All right, and the only difference is whether the phospho side is on this serine or the phospho side is on this threaning. I want you to raise your hand if you think it's on the threaning. Now I want you to raise your hand if it's on the serine. You have to pick one. Come on, come on. Okay, serine? Okay, who else wants to go serine? Anybody who doesn't vote doesn't get the lunch coupon tomorrow. All right, so the answer is yes, there's a serine. And you should have been able to vote, okay, because you don't have to know anything to see that when you look at the labeling, there's something that's not assigned here and it is assigned here, okay. All right, but let's talk about what is assigned and why, okay. So the fundamental premise of being able to pick where the thing is is you have to have fragmentation between the possibilities, all right. So in this case, you have this single ion right here in the spectrum, which can be interpreted as the Y7 ion for cleavage right there, where 101 would be the mass of threaning in its unmodified form. 167 is the mass of serine, which is 87, plus 80, which is the phosphate, okay. All right, so if you were instead to allow 87 for the threaning or for the serine, that would stick this in here in a sort of messy part of the spectrum. And then the gap out here would be shown as this ion to this ion and then that would leave that unexplained, okay. But because those two residues are right next to each other, you're not gonna get much information to work with in order to make your decision, okay. So in cases like this, and it's gonna be often the case that if you have to determine two choices that are right next to each other, you're gonna have to make that decision on maybe one or two peaks in the spectrum, okay. All right, let's talk about the range of possibilities now that could happen here, okay. If you have, if you're looking for phosphorylation sites, the precursor mass is 80 Dalton's higher, so you know you've got phosphate, and then you look at the sequence, can I, what's going on, I don't, what's here, okay. All right, if you look at that sequence, there's only one serine, threaning or thyracine in it. So you don't even need to look at the mass spectrum to figure out which one is labeled, or which is phosphorylated. It's gonna be that one. All right, I am having, I'm gonna switch to the pointer here, okay. It takes a lot of time to figure that back to getting the red spot on the thing, so I'm just gonna switch, okay. So in this case, you will have a peptide sequence where you have a serine or a threaning, and so you could, if you have enough information, you could confidently say that the phosphate is on the serine, and we would call that a 99% chance of being correct, okay. Let's suppose here you have one phosphate, and it could be on any of these three serines out here. If you have fragmentation between them, you can tell the difference, okay. And I'm gonna show you a spectrum where there's fragmentation between serines two and three, so we can say it's not on serine three, but we can't tell the difference between first and second serine, okay. When you get multiple phosphocytes in the same peptide, that gets a bit trickier, and this is illustrating all the possible places, the combinations that you could put them, and then I'm gonna show you a spectrum that gives you the ability to tell that there has to be one on this serine, not on that threaning, but then the second one we can't tell where it is, okay. So this is how complicated this kind of stuff gets, and when you are doing proteogenomic work, and you wanna look at the phosphodata set, and you look at the list, and you're like, there's all these things in this list that don't have clear assignments of the serine threaning. Well, that's a feature of the data that you gotta deal with. One of the ways you might deal with this, throw out everything that's not confidently indicated to a particular position, okay. Here are the spectra that give you the cases that I just described, okay. So here is a spectrum where we can confidently put the phosphate on the serine, and these ions in the spectrum, Y5, 6, and 7, are separated by the right masses, oh, they should have been labeled, okay.  So that can place the phosphate on that serine, not on that threaning. This gap over here is gonna be 101, okay. Here's the example where the Y13 ion, Y13 doubly charge right here, allows us to fragment between the second and third serine. So we know that the third serine now is not phosphorylated, but there is no fragmentation between the first two, okay. So we can't tell where that is. All right, here's the complicated one where there is two phosphorylation sites. The precursor mass is 160 greater than the unmodified version for this sequence. We have a fragment ion, Y9 and 10, that gap there is 187, which is gonna say that that's phospho-serine, and then Y5 and 9 here, there's not very good fragmentation between those, and so we can't tell where the localization is, okay. All right, so when you're gonna write something, so I tried to show you graphically, this is when you look at the spectrum, can you have the information? If you're gonna write a program to do this, right, these are some of the things that you got to put into the design of your experiment. You're gonna think about all of these things, okay. I think the most important of those things are shown here. The choice about how you decide what peaks are gonna be used to make your decision, and then how do you clearly represent the certainty or ambiguity in the localization decisions that the program has made, okay. There will be different choices made by people that write the programs about how to deal with the rest of these issues, okay. And then today, there is not a universally applied way of determining a false localization rate from these scoring things, whereas the target decoy calculation for identification is practiced throughout the field, okay. All right, this is one of the first automated scoring approaches and it is again using this binomial probability theorem, but instead of using the calculation based on all the possible fragmentation of the peptide, it's limited to just the fragmentation between the sites that you're trying to distinguish which have the localization, okay. But otherwise it uses the same framework, the same mass accuracy assumptions. And when you get down to the, what score threshold you're gonna use, it comes down to essentially saying that there has to be two good peaks that meet the scoring threshold, okay. At the time that this was published, the authors used a particular score threshold, to forget exactly what the value was. And then like a year or two later, they decided they could say they have more identifications if they made the threshold lower, okay. And it was essentially by saying instead of two peaks, you were gonna allow one peak to make the decision, okay. All right, but you have this nice descriptive way of using a mathematical calculation, okay. When I wrote the calculation, I've tried to think of it more intuitively and I calculate the score difference in the identification scores given various possible places and the decisions I made were on the quality of the information that gave those score distinctions, okay. I said that I want the ion type that you allow to make the decision has to be one of the highest information ion types. It's gotta be a B or a Y ion. You're not gonna make the decision based on one ion that's a B minus water ion. You're also not gonna make the decision based on a tiny little peak that could be mistaken for noise, okay. So what I sought to do is say that it's gonna be a B or Y ion and the relative intensity has to be at least 10% of the base peak. So it's a solid peak, it's not noise. That works out to giving a score threshold that's of 1.1. In conclusion, we hope that this lecture and the series of five lectures so far has helped you to appreciate the importance of sample preparation for mass spectrometry-based identification of peptides. The need for enrichment of post-translational modified forms of peptide prior to MS analysis. The lecture has also provided you the glimpse of how impurities in the sample can lead to the errors during the identification of peptides. And additionally, you are introduced to the concepts of PSMs and how a specific software like Spectrum Mill uses PSM to score the hits. Lastly, you were explained the concepts of phosphor site localization and scoring using suitable examples. In the next lecture, Dr. Karl Klauser will conduct hands-on sessions to help you interpret the MSMS spectra manually. Thank you.


I'm James Swanick from the Alcohol Freedom Formula. There's some free resources for you down below to get started on your alcohol freedom journey.  There's zero nutritional benefit to it whatsoever.   Here's the thing, it's not just the money that you pay to buy the alcohol, it's also the lost opportunities because you're drinking alcohol. Do you have a dream for your life? Do you have a business idea? Do you want to get a promotion at work? If we look at it from purely a financial point of view here, let's just say that you really want to start a business, but you're drinking alcohol and that is keeping you numbed out. It's not inspiring you to take action. You know you should be doing it, but you're procrastinating because you're in these days of numbing yourself with alcohol. Well, what's the opportunity cost there? You could have started the business already and already have generated tens of thousands of dollars, maybe even hundreds of thousands of dollars, but you haven't because you're using alcohol to numb yourself. So it's fair to say that even though you may only be paying a certain amount of money to purchase alcohol, the effects of that is keeping you numb, keeping you stuck in procrastination. You're not taking action and because you're not taking action, you're not generating revenue for yourself from a business that you might create. Likewise, if you have a job and you can climb the corporate ladder, so to speak, or you can get a promotion, or maybe you're in sales, for example, and how much money you generate for yourself is directly proportionate to how effective and productive you are in your business. If you are drinking alcohol, I already know you're operating at only about a six out of 10, because A players do not drink alcohol, B and C and D players do, and they stay in this lethargy, this fogginess, this muddy brain where alcohol is always like playing a part and you're always playing catch-up, where you're tired and lethargic the day after, even if you didn't drink a huge amount, even if you had one or two drinks, that's enough to disrupt your sleep, which means you're not as productive and effective in your job the following day. Now, if you're operating at a six out of 10, let me give you the benefit of the doubt, even if you're operating at a seven out of 10 in efficiency and productivity in your work and you get paid, let's say, $70,000, how much would you get paid if you were operating at a nine out of 10? Would you get a 20 grand a year raise, a 30 grand a year raise? Because the difference between a six or a seven out of 10, when you're tired from drinking, and a nine out of 10, when you have clarity and energy and focus and productivity and efficiency and strategy and you make things happen, could potentially be tens of thousands of dollars. If you're a business owner and entrepreneur right now, and you're doing it and you have a business that does say a quarter of a million dollars in revenue and you're operating at a six out of 10, you know when you're operating at a six out of 10, and you know that your business could double its revenues. This year, if its leader, if its owner, i.e. you, was at the top of his or her game, operating at a nine out of 10. So your alcohol is costing you in that scenario a quarter of a million dollars in revenue each year. You see, it's not just the money that you hand over with your credit card or your cash. To purchase the alcohol, it's the lost financial opportunities because you're not doing what you know you should be doing.   Now, you might have a business and you're like, how do I avoid paying tax? Well, how do I reduce my tax liability? You might go to a clever financial advisor who gives you all these great strategies and you may implement them and they're terrific. They're great. Do that.  It's keeping you unclear. It's keeping you unmotivated. It's keeping you unenthusiastic. It's keeping you in a state of mediocrity, of weak or average performance. Are you a B player or a C player or do you want to be an A player?   It's great that you save tax. Maybe you got an accountant, maybe you do your taxes yourself, maybe you're in a job and the taxes get taken out automatically, which is great. Maybe you don't even have to think about that.    Wake up. Understand that there is a cost to your drinking, not just the cost of purchasing the alcohol, but the lost opportunity cost. What have you missed out on? How much money could you have made if you were operating at a nine out of 10? How much income could you have generated for yourself if you were clear headed and not foggy, if you were energised and not lethargic? If you had strategy instead of muddiness? What's it actually cost you?  Stop it.  Give up the booze. It is a waste of time and it is keeping you stuck in mediocrity. Go ahead, post a comment down below. I want to hear from you. Share this video now with someone who you know is drinking a little bit too much. Like it, share it, comment, and stop paying the dumb tax.