Very nice. Ah, there you go. Thank you. Thank you very much. Very well. Thank you now. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Good afternoon, honour guests, ladies and gentlemen. On behalf of the main REH rural fibre gate I would like warmly to welcome His Excellency the Governor-General of the Commonwealth of Australia, General the Honourable Sir Peter Kosko, I would also like to welcome the Honourable Kim Wells, Member of Parliament, Minister for Police and Emergency Services and Minister for Push Fire Response. I wish to give a special welcome to all those here who have been our sponsors, generously donating amounts large and small that have been crucial in building and equipping a station that can make all our needs, making the generosity of the Government of the CFA even more effective than the final product. Lastly, I wish to thank the members of our community who have come along today to support us in this new beginning and to take pride in the partnership that makes an effective CFA of local volunteers working with the community. Now, with great pleasure, I call upon the Governor-General formally to open our main bridge fire station. I would like to thank John's acknowledgement of the ancestral traditional owners of land, the Grinron people, my own respects and pay my respects to their elders, past and present and to any other elders of other nations or tribes who are here today. I thank John and Ann for their welcome outside, their hosting today. It's really a joyous and important occasion, joyous for me and that I get to see a community expressing itself in that great volunteerism which characterises Australia. The volunteerism, of course, through the main bridge rural fire brigade where so many of the people who participate do so as an additional parcel in their lives, it's not the main thing they do. So from time to time it might well be the most important thing they do. But the volunteerism I'm talking about is the philanthropic efforts such as you'll see on the back of this program. Scores, probably hundreds of names in there from individual philanthropists to businesses and they describe a geographic area which is of course amongst the main bridge responsibility but as we know, philanthropy knows, no geographic boundaries and I'm very sure some people from far afield have what I'm sure they contribute. All of Australia holds its breath during bushfire season and it's not long before somewhere or another in this great wide round land fire comes to ravage, hold communities, rural economy, putting lives, livestock and livelihoods at risk. Like so many other Australians when those terrible bushfires occurred in another place but not so different to here, not so long ago I was horrified at the scenes of devastation and the impact of fire and its ravages on people's well-being, their optimism for the future. But like all of you, I was uplifted to see those hundreds, indeed thousands of people who rally to stop the fires, to divert them, to save property, to save livelihoods and of course to save lives. And like you, we're all impelled in the aftermath to dig deep, to do whatever we could, not only to help those whose lives have been so drastically affected, but to show with the same fortitude in a different way the sort of resolution, determination, indomitable spirit of the firefighters who put their own lives on the line and who will do so every time there is a need. I was at a memorial service in St Patrick's Cathedral in Melbourne in the aftermath of the shooting down of the Dutch aircraft, of the Malaysian aircraft over the Ukraine. And at this memorial service we were extending our condolences to all the bereave, the vast majority of whom were in St Patrick's. And in amongst all the solemnity of that service there were two songs that were sung in a war modern style, they weren't hymns, they were done by some brilliant young singers and string quartet and a person on the guitar and another on the keyboard. And if you're watching the service you might recall, I won't describe the first song, it was beautiful and very evocative, but the second and last song sung right at the end of the service was a version of the very popular Australian ballad, I Am Australian. It was a version of that ballad that was done in the aftermath of the Victorian who's fires. And it was done in slow tempo and it was replicated there in the cathedral, sung by a lady called Katie Noon beautifully. And the last line I think epitomises the Australianness that is represented by the CFA, by the Royal Fire Brigades, by people like the Main Ridge Royal Fire Brigade. And it's finished on this note, it says, in that one which would finish, I Am You Are We Are Australian, it said, you're not alone, we are with you, we are Australian. And that says it all, and that says what we are here today. To inaugurate, to celebrate and rededicate ourselves as a community, and these men and women who will operate right at the forefront of the protection of the community, come the inevitable next time they're called to save lives and property as a result of a bush fire. I had a prepared speech here, you just heard what I wanted to say. I think it's a magnificent facility, nothing less for the people who will need to operate here to store their kit, to order, to house all of their equipment, to train, to turn it up for training and to come here before and after operational missions to fight fires. I'll come here to get their new orders, to refresh themselves, to be given provisions by the indomitable people such as the CWA and then go out again. They deserve nothing less than this magnificent facility. So it's now with great pleasure I declare the Main Ridge Volunteer Royal Fire Station open. For that introduction, His Excellency, we are deeply honoured to have you here today. I'd also like to acknowledge our other special guests today, Kim Wells, Minister for Police and Emergency Services, Martin Hickson, Minister for Education. What a wonderful day, an exciting day for Main Ridge Fire Brigade. And it's my pleasure to be here to witness this official opening of this new station. The Main Ridge Royal Fire Brigade has a long and proud history, having formed in 1945 and served its community faithfully since that time. Back in those days, the only equipment firefighters had was NAPSAC sprayers and Army bootbeaters. Thankfully, firefighting has come a long way since then. With this 21 strong member, Volunteer Brigade, continuing to provide a vital service to this community. Today, this small but strong Volunteer Brigade serves its community faithfully responding to a range of incidents including fires, car accidents and other events. Over the years, the Brigade has responded to fires in this area. The most recent was the 1997 Arthur Seat Firewood Strait in the Homes. But as His Excellency mentioned, this Brigade also provides vital support statewide when there are major incidents. And that included Ash Wenders Day and more recently Black Saturday Wing Crews from this Brigade assisted with the fires around Ariesville and King Lake. This new station we're opening today sits opposite the site of the original Main Ridge and is the third station local firefighters have utilised. It is more spacious and both three engine bays with drive-through access, car parking, an office, multi-purpose room, kitchen. And why don't I add toilets, because that's one of the things when you've driven a long way to come here that you look for. I'm always pleased to find toilet supply stations. I also offer our thanks to the government for their financial support in helping to build this new station. There are a number of new stations that have been building this for you here in and we offer our thanks particularly to the government. This station has cost $789,000 and I too would like to add my thanks to the local community and to the Brigade itself for their fundraising that's already $1,000 raised to help make sure this was a first-class facility. I'm confident this new fire station will further boost Brigade's firefighting capability and see them protect the community for many years to come. To all CFA members present today, I thank you for your invaluable service and I hope you enjoy your own station. Thanks also to the families who give you your support. Lastly, we are entering the fire season. A day like today is a stark reminder of just how quickly summer has come upon us even though technically it's still spring. Please place a high value on your safety through this season. Look after yourself and your mates. Thank you very much. Thank you Mr. President. And now for one of the heroes of the hour without whose vision, sheer persistence and boldness we would very likely have neither this station nor the honour of having it open by no less than our nation's vice-legal head of state. I call upon Mainridge CFA Captain Dian Trapeit to respond on behalf of our Brigade and our local community. The new staff boy will be able to speak this, but I'll make it up at my go. Good afternoon, Your Excellency. Other distinguished guests and members of the Mainridge community. What a wonderful day it is for Mainridge to have this wonderful facility and to have it open for the Excellency, Sir Peter Cosgrove. And I thank you very much for you accepting my invitation to come. I would like to thank the Shire. We put a proposal to the Shire about three years ago to obtain this parcel of land to build our new station on. If it wasn't for our councillors who all voted unanimously that we could have had this parcel, we would have still been on the old site. And it's a wonderful piece of land and I'm just so very grateful for the Shire for their vision and foresight to give us a parcel of land. Now, just to give you a bit of background, which has already been done, but after Gluck sat down, the government decided to give 140 new stations out around country Victoria. We had some wonderful people within CFA who fought long and hard to get Mainridge a new station. And I'm glad they fought so hard because this is the evidence of their work. So without those workers and those officers within the CFA, this would not have happened also. So thank you very much. I don't want to go into naming each individual one of you, but you know who you are and we are very, very grateful. The CFA had about 59,000 volunteer, 5,000 gay people, 1,800 permanent staff, and they have 1,200 odd stations. So we are very lucky to be part of a very well-organised and very professional organisation of the CFA. It's not roughly what my speech is, but I'm living a little bit. I practice it at three o'clock this morning. Next Saturday. And I'm a bit hoarse now because I was hunting it out. At three o'clock, there's no one there to have a chook stone to give what I said. I'm also very grateful to all the local residents here in Main Beach who donated. If it wasn't for your contributions to our extension, half of you guys are sitting in the part we paid for and unfortunately, the other half are sitting in the government side. If it wasn't for your support, this would not have happened and it is a station which will give us 50 years plus of wonderful facilities to size. We have issues with truck size. Every year trucks get bigger, stations tend to get smaller. So we have so much room in here for a future expansion if we want different firefighting times as well. The better the equipment, we have the room in which to put it in. So I am very grateful to all the local residents who have supported us, which lead me onto the community groups. All the charities, the service club, the corporate sponsors have been brilliant. They have given us so much extra which allows us to have the nicer things within the station. So if it wasn't for the corporate people, also this has been very humbling to think how much they have given from such a wide scope of people.  It's a month earlier than normal. So go home, clean up, prepare. They like the driveways. They'll have the trucks to come in.  And if you're going to leave, leave early. Don't wait for the death knock because it makes the road terribly congested. So fire plan, prepare and leave early to need to. And my final word is have a happy and safe fire through Christmas. And thank you very much.


who, Makoba, are you in the room one second? Yeah, Makoba, you're in the room. Could you, because I know there was a question that you've had with regards to inflation and I think it was inflation in GDP. Makoba, are you there? Yeah, right. So I don't know if you can unmute your mic and ask the question because I'm talking about the economy and inflation right now. Hopefully I can explain your question within a few minutes or so. So what was it again? So what was your question? Hi, Makoba. Yeah, I can hear you. Yeah, yeah. The problem is when the inflation and GDP is going in two directions. Maybe for example, if the economy is into recession, it's going into recession and the inflation is going up away from 2% target. Yeah. I get confused what central bank gonna do if there is a difference in, because in order for the central bank to rise rates, GDP and inflation, they must be going in the same direction. They must be going high. So the case when they are not going in the same direction. When I was trying to answer the test, I faced some situation where the economy is in contraction. At the same time, inflation is going high. So it's just a problem for me to understand what the direction. Okay, all right. So what you're describing and thank you for that as well. And it is a difficult question to ask to answer. And it's what's known as stagflation, Makoba. Yeah, so stagflation, yeah, is, let me just type in stagflation, all right, the definition of stagflation. And it's when in economics, zoom in, so in economics, stagflation or recession inflation is a situation where inflation rate is high, yeah, going higher or increasing, and the economic growth rate slows and unemployment remains steadily high, right? But what we're focusing on is this. And this is basically exactly what you're talking about. So we're going into economic contraction, negative growth potentially and slowing down, but inflation is going high. So it's known as stagflation, known as stagflation. And so it is what is a bit of a conundrum for central banks, right? And so I guess anyone who hasn't taken the test is probably gonna get the answer now. It's gonna get the answer for the test, but as long as you understand it, this is over one of the questions of the test, I should say, but as long as you understand it, that's the main thing. So it's, as you correctly said, typically for central banks to high crates, what you would expect to happen is for the economy to be growing, yeah? But this isn't always the case. We don't live in a perfect world, unfortunately. So there are gonna be situations economically and inflation-wise where inflation is going higher, but we have what is known as, I guess, a contraction or an economic slowdown. So as you're saying, what do you do in that situation? We're really kind of like two things or that you can do, yeah? Or what central banks, because really the question is, to think like a central bank, right? So what do you prioritize? Do you prioritize GDP, right? IE, you know, a recession, right? Do you prioritize that or do you prioritize inflation? What is the bigger threat? And it's difficult to say. I can't, you know, it's not a one answer. That's supposed to be an end, by the way. It's not a one answer thing. It's not a binary thing where it's like, if this happens, then we must do this. It's very dynamic in the way that you have to think about these things. But in terms of stagflation, yeah? There are, in the way to trade stagflation, there are really, you know, kind of two things that you can do, yeah? Is going into detail, is actually looking at what the market is more focused on in terms of, is it focused on the central bank? Is it focused on the inflation side of things because they have to get things back to the 2% target? Yeah? Or are they focused on GDP and avoiding a recession? Yeah? So in the situation that we're in currently today's, you know, not hypotheticals, but in today's environment, we've known for a long time or for a while that central banks are prioritizing inflation over GDP. Hence the reason why they are hiking rates, even though you're having an economic slowdown, we're going into the contraction, potentially, you know, recession phase, if this is recession, this is the contraction phase, yeah, of the economic cycle and that's maybe the bustle slump and then that's the recovery right there, yeah? So what they're doing is, is they're saying, okay, before we potentially get to recession, before the data gets to a recession, right? We have a bit of leeway. Let's try to combat inflation first by hiking interest rates, right? By hiking interest rates, yeah? To try to get inflation back down to that 2% target. Yeah? Because inflation at the moment is, you know, is basically getting out of control and I'm going to talk about that, you know? I think the data come out today, which was talking about inflation at 9.1% in Europe. Is that correct? Something like that? Someone double-checked that for me, but I think it's over about 9.192%. I think I updated the fundamental analysis spreadsheet. Europe is actually number three on the fundamental analysis spreadsheet. Crazy to think. I haven't seen that in a long time, but I'll get to that anyways in a little bit. But to understand what, you know, ultimately central banks are thinking, the central bankers are thinking about, you know, stack inflation, it's either one of two things. Either they're prioritizing GDP or they're prioritizing inflation. And we've known that they've been prioritizing inflation because, so Daniel says 9.1% and core is 4.3. Okay, brilliant. They've been prioritizing inflation, right? So hence the reason why you're seeing interest rates, right? Hike, you know, hike interest rates. Now that doesn't always mean, it doesn't always translate into a currency being appreciating because a central bank is hiking rates. Typically we know that to be true, yeah? We know that to be true. Or what typically happens historically is that whenever central banks hike rates, they obviously, you know, that should appreciate and create demand for a currency because, you know, investors who are holding that currency want a better return, right? So, you know, that's basically what it is. But when you have a recession on the horizon or potential recession on the horizon, you know, the question is where do investors, what are investors really concerned about? Are they more concerned about the coming recession, the impending recession?  On their investment by holding that particular currency. It's very difficult to know, to get into the minds of, you know, millions of people, right? It's difficult to do that. But so the general consensus is, or I should say really, is understanding the nuances, yeah, of every situation, because every situation is unique. The last stagflation situation, there would be, there are different, there are slightly different factors to what is happening in, you know, 2022. And, you know, if we ever have another stagflation scenario and maybe the next 10, 15, 20 years, it will be slightly different to how what we're facing today, yeah? So to kind of wrap this up, what does that mean for, for how you trade currencies? Because ultimately that's what, you know, the question that we're ultimately asking. Now, if you're unsure about stagflation, personally, you can either stay out of that currency, yeah?  But as we trade currencies in pairs, yeah? The smarter thing to do, I guess, would be to trade a currency, yeah? Because currencies are obviously traded against each other, trading them in pairs, is to trade, is to buy a currency that is, is doing economically good, right? Versus a currency that isn't doing, or that is in stagflation, right? So if that's in stagflation and there's a lot of uncertainty around, yeah? Where do you think the majority of traders or what should follow logically, yeah? In terms of where would you put your money into? What we, where would you invest is, would you invest in an economy, in a country, in a central bank that is more stable or would you go into stagflation? You know, risk your money in a country and an economy that is going into stagflation, right? It's a no-brainer to put your money into, you know, this economy, right? So if you can identify, which we do, the economy, yeah, that is doing, you know, pretty well or doing okay or again, you know, the phrase that I use all the time is the dog with the least fleas compared to a country that is, you know, suffering from stagflation and a lot of uncertainty, this is where you wanna buy and this is where you would want to sell, yeah? And if you're obviously buying the base currency and you're selling the quote currency, then you should see the market do something like this. If this is, you know, if you're buying a quote currency, let's say for example, that is the economy that you think is doing well and that's the economy that is in stagflation, then you will see or typically see a downtrend. I'm saying downtrends are gonna, you know, look perfect and they're gonna make perfect lower highs and lower lows, right? But ultimately you should see the market do that. And you'll see in that take place. And I think Spenk, yeah, Spenk said that's, you know, he mentioned the UK, right? And the reason why the UK is going through, it's, you know, it's selling off is because there are a number of risk factors as well as stagflation going on with the UK economy, the cost of living crisis, inflation going higher. And that's the same across the board, right? Same in Europe and it's the same even in the US. But again, when you compare all three or all, you know, eight currencies that we trade side by side, which is what we're looking at in terms of the fundamental analysis spreadsheet. Again, it's all about who is the best of the worst over time.  So I can't say this day in time or this week in time, this is exactly what's gonna happen. No one can.  On that one trade, nobody knows. So it's a probabilities game. So I know overall, and you guys have been with me for at least a minimum now of two to three months and you've seen for your own eyes, right? Week in, week out, the fundamental analysis videos I've been doing week to week and you're seeing that play out in the market, right? Overall, yeah, when we've looked at when you joined and I've been saying, okay, you know, short, for example, pound dollar over the last two to three months, what's happened to the pound dollar? Where was the money really being made to the short side? And it's because the pound dollar, yeah, the dollar CAD and I can name, you know, loads of currencies that you guys have been trading them, where my bias has literally been either long or short and you're seeing that play out. So that is really how you trade stagflation, yeah? But it is understanding the nuances you do have to keep abreast with is what the central banks are actually saying, what they're prioritizing and what's going on in the country, what other risk factors do they have and what the market thinks of those risk factors? And we'll get into confirming that with bank analysis and things like that. But does that explain your question, Makoba? Does that answer your question? And has anyone else got any questions that they want to ask about that? Is that clear for everyone? Yeah, 100% survey, brilliant, all right, cool. Yeah, very clear, excellent, excellent. So anyone who's new or doesn't understand these things, brilliant, that's how you trade stagflation. Yeah, you're not trading it in a vacuum, it's not in isolation. Trade it against the currency that is, you know, that is doing better than that currency and then it's easier to see. Yeah, excellent, okay, brilliant. Right, let's move on. So with that being said, going back to Jackson Hole Review. Yeah, so pretty much all central banks are hawkish. You know, we've got Jackson Hole, top central bank has delivered a hawkish message to Jackson Hole and ECB's officials as well that are acting forcefully to curb red-hot prices. So that is, you know, pretty much the nuts and bolts of Jackson Hole, right? And again, we've seen that kind of playing out. And I think the only bank that was pretty much pledging the loose policy, meaning that they're not looking to high crates. And by the way, the Bank of Japan does have a habit historically of surprising the market as well. So you have to be aware of that. You know, it's like the Bank of Canada as well where they might say one thing and then, you know, maybe, you know, two months down the line, all of a sudden they just switch their bias, right?  But it sets up a nice trade when you understand, in, for example, with the Bank of Japan, we all know, or we shouldn't know anyway, that the 140s, yeah, 140, 141 area is the area where you wanna look, where they basically said that they may start to, you know, change policy or adjust the policy in terms of, you know, maybe interest rates, you know, adjusting their bonds, quantitative easing, et cetera, right? So 140 is still the level to kind of, you know, pay attention to. Now, if you still stop, if you stop to see prices go above that 140, I'm not saying that it's gonna be at that exact price, right? Nobody knows, but if it starts to go around the 140, but you also start to see, yeah, that inflation for Japan is rising, again, away from their 2% target, because I think they're at 2.4 or something like that, 2.6% inflation, and you start to see maybe it goes to maybe 3%. The central banks, you know, the Bank of Japan are gonna be forced to do what? A hike or cut rates? Are they gonna continue to, you know, be dovish or would they start to turn a hawkish? That's the question. As Sabi says, hike, so hawkish, Ademola says hawkish, yeah, that's exactly it. Daniel says, hike, absolutely.  So what potentially could happen? So although right now they're quite dovish and continues to be dovish, that could change depending on obviously inflation and inflation being driven up by a weaker yen, right? A weaker yen and a devaluing yen pushes up inflation, which then forces the central bank to have to potentially now step in to try to get inflation to come down because they don't want inflation to get to four, five, six percent, seven percent, et cetera. Yeah. So there's that. So again, no matter what guys, if you have a solid understanding of how fundamental analysis works, you can, I'm not saying that you can never be taken off God. Of course you can, but you have a roadmap and you can have a plan for the future, right? And you know, you can understand why bankers will be doing certain things. And on many, some occasions you can actually get ahead of, you know, the situation and start to position you.


Okay, so I think we've dealt with our question as we'll dive into metabolite identification and quantification. So we're going to look at, essentially, and that's to pick up where I was emphasizing last couple slides from the last module, which is quantitative metabolomics. We call it targeted, it's also untargeted, but it's still quantitative. So we're going to learn about spectral deconvolution or assignment, and we're going to use it both for NMR, for GCMS, and then also LCMS. And then we're going to look at a very variety of mass database searches and databases. So the goal in metabolomics is to go essentially from spectra, whether it's GCMS, LCMS, NMR, whatever, to lists of compounds and their concentrations. And the concentrations can be relative concentrations or absolute. My own preference is to have absolute concentrations, and that's certainly a growing trend in the field of metabolomics. So historically the problem with metabolomics was it was lagging behind the fields of genomics and proteomics. If you had a genome sequence, you could very quickly either translate the genome to protein sequence or proteome sequence, and then you could just do a blast search, and you could basically identify all of your genes and proteins. Sequence alignment, sequence comparisons could identify functions, the names, all kinds of things. And this is something you could do in a matter of minutes. In the world of proteomics, if you've got MS spectra and MS MS data, you can submit your unknowns to the mascot server and very quickly get protein identifications. So software, public software has existed both in the world of genomics and proteomics for a long time to identify your unknowns.  In the world of metabolomics, for a long, long time, there just wasn't that tool. Basically people would go to the library, pull out books published in the 1960s and compare their spectra to whatever, and then hope that they could find what they were looking for. And so that's been an issue with metabolomics and probably one of the reasons why it was the last to sort of join the triumvirate of omics. We're going to essentially in the next bit show how that's now changed and show you some of the tools to do that. But I think there's also this issue of what's now become the unknown, unknown problem. It's partly picking up from Donald Rumsfeld, if people might remember his quote. There are known unknowns that say we know, there are some things we do not know, but there are also unknown unknowns. The ones we don't know, we don't know. Anyways, in the case of metabolomics, there's lots of situations where the peaks are there, and so the moment we see them, I can't really tell from a retention time or even just a collection of mass, M over Z values or chemical shifts what the compound is. But by submitting it to a database, these databases or software tools actually can identify them because they were previously identified. They sit in a database, so they've gone from being unknown to known. But then there's the more challenging ones which are compounds that, yes, you've identified or you've collected spectra from, but they match to nothing in any of the known databases. The mass doesn't match to anything. The NMR spectrum doesn't match to anything. The retention time doesn't match to anything or a combination of all those threes. And so in those cases, for the unknown unknowns, you have to actually use computer aided structure elucidation or case methods, which are something we'll briefly talk about. But what we're looking at is mostly the known unknowns. That is, there's some point in history has actually identified, purified the compound, collected the spectra for it. And so it's your task really just to see if it's in this particular biosample or not and what its concentrations may be. So for those known unknowns, we do either something called spectral deconvolution or spectral assignment. And so you're doing, all you're doing is matching peaks to a known set of peaks from pure compounds using a pre-compiled database. So someone's had to collect these pure compounds and run literally thousands of spectra, tabulate them, put them online or in books, and make them searchable. But that's been done and it's being actively pursued and it works for NMR, GCMS, LCMS, and MSMS data. And so that identification of the known unknowns is what I call quantitative metabolomics. People can call it targeted, but it's also untargeted because in the case of NMR and GCMS you're not really targeting. So that's the theme for today, at least for Module 2. So in the case of metabolite deconvolution by NMR, this is essentially what you're doing. If you're given a mixture, which is at the top, and if I had shielded off the other components, the question is, you know, is that mixture one compound, two compounds, three compounds? If you look at it, there's I think six spectral clusters and say maybe it's six compounds. Or if you think in the world of GCMS, you might say there's 20 odd compounds. Well, what you can see is in fact that top spectrum is the sum of three spectra from compound A, B, and C. And not only can you see that it's a sum, but you can also get an estimate of the concentrations. The compound A equals compound B in concentration and compound C probably also equals compound B in concentration. So there's three equal molar concentrations of three different compounds. In some cases, the spectra peaks overlap. They have exactly the same chemical shifts. In other cases, they are unique. So this is relatively simple to do with three compounds in a simple mixture. It's much more difficult to do with 50 or 100 or 1000 compounds in a complex mixture. But that's the challenge. Now, there are tools and software programs that actually allow you to do this. There's a company called Kinomics, which is based in Edmonton, which allows you to do spectral deconvolution with NMR. And the software itself is something you can download and run it for free, at least for a month, I think. So it allows you to manually process the NMR spectra. So this is where you do the Fourier transform. You convert what's called the free induction K into an NMR spectrum. Then you phase it with the software that they have. You can remove the water signal, which is usually very, very prominent in an NMR spectrum. It'll perform baseline correction, which is something you have to do with MS and LC and all kinds of things. You also have to do with NMR. It'll do some referencing so it identifies the chemical shift. It'll normalize the peaks. Again, you have to do that manually. And then you fit the spectra. So you'll take the reference spectra and you say, I think this peak looks like, you know, loosing. So you pull out the loosing peak and it's done with a mouse click and it will fit that. And then you can shift it around. And it's actually very easy to learn and to use. And in past years, we would actually teach people how to use that. But we found that it was taking a long time. And in fact, the tendency is that it takes about half an hour to even an hour for a person to fit a spectrum that's maybe relatively simple, but has maybe 40 compounds. And if we had 30 of you doing the fitting, we would get 30 different fits. Even though it's the same compound, same mixture, same solution, just the tendency for people doing different styles of baseline correction, different styles for fitting and phasing, all of which can lead to misinterpretation. However, there are other tools. So Brooker has a software system called Amix, not really as friendly or as usable as the Kinomics one. But there's some automatic tools. Brooker has developed an entire NMR system and software. It sells for about a million dollars for analyzing juice. And then you can get another one that'll analyze wine. And these will automatically identify the compounds in juice samples and in wine. And there's about 40 compounds that these can identify. There's a tool developed by Tim Ebbell's group at Imperial College called Batman, which uses a Bayesian method to automatically assign or characterize NMR spectra. And Batman typically will take about 8 to 10 hours for a sample mixture of about 20 compounds. So it's not exactly fast and not really amenable to a course like this where we've got 30 people. So what we'll talk about and show you in the lab is a tool called Bazel, which was developed in my lab in collaboration with Russ Kreiner in Computing Science at the University of Alberta. Why do we want to do automation? Well, it's fast. So instead of 30 minutes a sample, you could potentially do it in one minute per sample. And it's consistent. So it'll give you identical results regardless of what sample is done and it'll do exactly the same all the time. So if it's wrong, it'll make the same mistake every time. But if it's right, it'll do it correct every time. Obviously, if you've got hundreds or thousands of spectra, it's something you can leave on your own. You don't need teams of people staring at spectra or working on computers night and day, which is actually happening. And because it's a computer, it can also see signals that sometimes are not obvious to us unless you're a real expert. So there's one of the real strengths or many of the real strengths with automation. Yes. Generally, what you want to do with the Kinomics software is start with the original FID. Because the processing that Kinomics expects you to do, it guides you through that. And it actually does a really nice job. In fact, it generally does a better job than the NMR instruments themselves. It has much better baseline correction, much better phasing and water suppression than the instrument software. It's just best to take the FID, and that makes the FID somewhat independent of what someone else has done to it. Yes, this is all 1D NMR. There are some tools that allow you to do analysis with 2D. And the problem with 2D is it's not quantitative. So it's good for identification, but peak intensity is very considerably with 2D NMR. So that's why we're focusing on 1D here. I mentioned Batman, and so you can download the program, you can install it. Jason here has spent much of his life trying to install it and trying to run it. But some cool ideas, it's just incredibly slow. So we'll be using Bazel today. And what's nice about Bazel is it's actually web-based. So you don't have to download and install. You don't have to have an operating system. So all of you, I think everyone now has web access. So you should be able to do it. We've tested on many samples. We use it in my own lab, which is a core lab. So we've looked at hundreds of samples. It's quite accurate. It uses what's called probabilistic graphical models. And most of you have never heard of them. It's like a hidden Markov model, which some of you might have heard. And these are things that are used commonly in speech recognition. So if you've ever used speech recognition like Siri or anything like that, those use or analyze spectra and look for identifiable patterns. And that's how Siri or other speech recognition tools recognize words. Here, we're just making it try and recognize compounds. But it's a similar type of problem. It functions in an inferential way. So it uses inference techniques, which is what humans also use when they fit and we watched how people would fit spectra. And so that's sort of embedded into Bazel. The reason why it works is, and the reason why it's successful, even for humans to fit complex spectra, is they have to know what the spectra is. So is this of blood? Is it of urine? Is it cerebral spinal fluid, saliva? Is it hemolymph? Is it plant sap? If you know what it is, you also have a good idea of what its composition is. Give or take about 10 compounds. And so if you have that prior knowledge, which is where this Bayesian concept comes in, then it helps with the fitting. It makes it a convergent problem. What we've also done with Bazel is we've made it fully automated in the sense that it automatically phases things. It automatically gets the chemical shift reference. It automatically removes water, baseline corrects, and then it does all the deconvolution. By taking that away from the human, it gives it consistency. And that's critical, especially if you want to move this sort of thing into the clinic, which is one long-term plan. If you can make things automatic, then you can move it into, especially general practice for things like screening, for industrial work, or for clinical work. So these are some examples of where Bazel has fit some compounds and spectra. The description of Bazel actually just came out a couple of weeks ago, so we had to submit the slides before that came out. But anyways, you can see how complicated things can be. But you can also see how well fit it is. You can compare the green line to the black line, or the red line to the black line. And this is what Bazel's able to fit. Here it is working on Cerebral Spinal Fluid. And in this case, the fit was a human. An expert took them about 45 minutes, and you can compare the red to the black. Lots of peaks. There's about 45 compounds. Bazel, on the web server for the same sample, would take about five minutes. If we were running in parallel or a really fast computer, it would take just a minute or less. So the website, and this is actually, I guess, the reference for the paper as well, is very simple. It was actually designed by Jason Grant, who's with us. So is there any problems? Blame him. And so there's sample spectra you guys will be doing in your lab. You'll actually be uploading spectra, and it'll be processing and analyzing these things for you. But it's an example of, I think, a trend that's already happening in metabolomics, that things are moving more and more towards automation. Because, in some respects, why waste your time identifying compounds when really the interest is, what do these compounds do or mean? What's the biological interpretation? That's why you have your graduate degrees, or that's why you're pursuing your graduate degrees. So there is a trend, whether it's through software or through kits, to try and make metabolomics, at least the compound identification part, simpler, faster, more robust, more automatic. So, if you're operating basely, as I say, click on either example, and then press submit. And what you'll see within the first five seconds is it will take the, what's called the free induction decay, or FID, and do a Fourier transform of that. So it converts it into a spectrum. And that's what the spectrum looks like initially, which is really awful. So it's got peaks pointing up and down. So that's called the defazed spectrum. This is what a lot of NMR spectrum looks like. It has a giant water peak, which covers all kinds of things. That's the thing in the center, because we're collecting the sample in water. So that's after five seconds. About 15 seconds later, it will have started phasing. And so now the peaks that were all upside down are now right side up. And that giant water peak that was sort of giant unfazed thing is now mostly phased. And so it's starting to look like a pretty good NMR spectrum. And then about 30 seconds later, it's now got a completely flat baseline. It's now properly referenced. The giant water signal is completely removed. So all the baseline correction, phasing, reference correction, peak deconvolution is now done. So about 30 seconds in all. And then over the next three to five minutes, it's going to try and identify every single peak in that spectrum. And this is what it'll produce. So you'll see, again, a spectrum, which you can use a variety of tools to zoom in. And it will display. And you can just barely see in the figure, there's a blue outline of what it's fit and a black, which is the actual spectrum. And I think you'll see that just about every peak or every peak is identified there. So five minutes, it's done what would have taken you guys probably an hour to fit using manual things.  It gives you a list of the compound, the HMDB identifier for the compound, the concentration for the compound, and the confidence score. So confidence of 10 means it's absolute certainty that the compound's there, nine, very, very certain. Five says we're not that sure. It could be there, it could be not, so it might be worthwhile looking at it in a little more detail. And I think anything below five is considered probably not there. So it's a web server, so it can't do everything for you. And so what it currently allows you to do is to look at serum, plasma, and cerebral spinal fluid. And that can be from humans or rats or any mammal, probably even fish and frogs and lizards too, but that's because these have larger, the same composition or expected composition. It doesn't work for urine, it doesn't work for plant sap, it doesn't work for wine or juice. And that's because you have to collect the reference specter for that and it's not trivial. It doesn't work for every kind of NMR, so it doesn't work for 400 or 700 or 800 or 900. But it works for the common ones. If you are going to be using it outside of this course, you should be really careful that you're preparing the samples exactly as described. And this is essentially in any analytical technique you have to follow a protocol. And I think what we found when we put this on the web, almost immediately I'd say 90% of the people didn't bother to read the protocol and so they'd be uploading anything and complaining that it wasn't working. So again, it helps to read. So right now it's in a single spectrum mode. It's a privileged opportunity with a bulk spectrum analysis that Jason has spent a long time working. Again, if it doesn't work, blame them. So that's what we'll see in the lab. So I'm going to switch now to metabolite identification by GCMS, which is a little different. So in GCMS recall we separate by gas chromatography and then as I said what you'll find is that typically under any given peak, if you're lucky it might be a single compound but more often than not it may represent two or three or ten compounds. So what we can see is we've taken this little peak and then we blow it up and we can see oh it's actually the sum of three peaks and within each of those three peaks we kind of get mass spectra. And in some cases the mass spectra aren't exactly totally pure but hopefully they're a little pure than others. So you can kind of deconvolute which ones are belonging to the red compound which ones are the black compound which ones are the green compound. Once you've got those spectra these are the electron impact or electron ionization spectra. Then you look up a database and the database would have pure compounds. MS spectra collected under identical conditions 70 electron volt, fragmentation energy and you would compare and you'd try and match them. So if you look at the red one very carefully you'll see that it matches the one that's circled and the blue one matches the one that's also circled and the green one matches the other one that's circled. So now you've identified the three compounds. Even though they had basically the same retention index they had three different spectrums so now you've identified them. So as I say the reason why that's possible is because at least with the electron ionization or electron impact ionization the compounds fragment in exactly the same way if you put them under the 70 electron volt potential. So that's why it's reproducible. If you used 65 electron volts or 100 electron volts you'd get very different spectra and so nothing would be identifiable. So again, follow the right protocol. So what you'll typically see is with an MS spectrum you normally would just see the parent ion or the molecular ion. So that's the compound with its molecular weight plus one if it's extra hydrogen. Occasionally if there's a gas in there you may have an adduct forming so you actually can see a peak that's actually of higher molecular weight. In this case it represents an additional chloride ion that's been added. Even on the lower molecular weights closer to the y-axis you'll see a whole bunch of other peaks. Those are the fragment ions. That's where all the information is because with GCMS the resolution is very low. It's typically one Dalton so it's not enough to identify a compound by molecular formulas but the fragment pattern has all the information. Now the intensities don't tell you a whole lot of what the dimensions do. Now the other thing you also have to remember in most cases as well, GCMS compounds are drivitized so they'll have trimethylsilane or they'll have methoxene derivatives or TBDMS derivatives and sometimes they'll be decorated at two or three or four different places and so you will have increased masses and so instead of seeing an adduct you'll see a TMS derivative or a double TMS derivative or a triple oxene derivative, whatever. So the masses that you'll get from GCMS and the perinion don't necessarily correspond to the pure original compound they correspond to the drivitized compounds. So for this one compound actually had six derivatizable amine or hydroxyl or acidic groups you could end up with six different groups or six different peaks actually all with different masses. So one compound, six peaks. So NMR is typically used to identify hydrophilic compounds water soluble compounds. GCMS is also pretty good at identifying hydrophilic compounds like amino acids very good with organic acids also very good with fatty acids which you can't generally detect by NMR or cholesterol in some cases. It has a mass limit so typically compounds less than 500 Daltons so you're not going to get to detect really big molecules or large lipids with GCMS. So I mentioned GC chromatography it's higher resolution, higher plates better reproducibility as well than LC. The other thing that's happened with the GCMS community is they've standardized everything unfortunately that hasn't happened with the LC or LCMS community so that still allows the EI spectra to be very comparable. When most people do GCMS work now is to take to use a combination of a software tool called AMDIS and a database called the NIST database of the National Institute for Standards database. So I'm not sure if NIST is up to version 12 or not but this is version 11 so it has hundreds of thousands of spectra of electron impact spectra. It also has other ion trap and Q-TOF and triple cod spectra but because things aren't so standard for those methods they're not that this database isn't as useful as the EI database. And there's also retention index values for about 20,000 compounds in this database. So remember what I said is that retention index can actually be used to identify or at least narrow down what a compound is. This is what the searching software looks like it's something that you download and install usually on Windows machine and you can see the mass spectra you can see compound lists you can see scores probability matches so it's quite informative and then you can see how the spectrum up in red matches the spectrum down in black which is again another visual cue about whether you've got a matching compound. So essential to how NIST works is this software tool processing tool called Automated Mass Spectral Deconvolution Identification System or EMDIS. So it does what Bazel does for NMR to some extent it tries to identify the background noise it identifies peaks so it does peak picking it does a spectral deconvolution pulling out the peaks the mass peaks from the GC peaks and then if you connected it to the NIST database it will do the compound identification using a scoring technique called the match factor or MF so it's measuring the similarity of the peaks of your query to the peaks in a database so it has a formal mathematical definition so it compares the intensity and the mass of each peak and it scales it by a factor of a thousand so it's essentially a dot product if you remember linear algebra and it's scaled so you get a perfect match perfect match factor will give you a thousand and some people will divide it to express it in terms of percentage and the rest is just a number from 0 to 1 but the way that the match factor officially was calculated is that perfect match was a thousand and imperfect match was 0 so if you're trying to do GCMS and you want to both identify and quantify and identify by retention index as well you need to have a bunch of external standards so usually the standards are L-canes and they usually span from octane to hexadecane and so this can be used to help determine your retention indices calibrate your retention indices and if you actually know the exact concentrations they can also be used to help calibrate a little bit with the quantitation generally you want to do a blank sample which allows you to identify peaks that might be rising from the solvent or the derivatization agents or stuff that's still stuck in the column so blank is really good to have and it's something that you often subtract the blank from your spectrum so you've got your calibration standards for your retention indices you've got your blank sample and then you run your sample of interest under the same conditions that you ran your blank so this is what your external standard for the retention index calibration would typically look like so you've got eight or nine peaks there you can see they're nicely separated and you now can scale everything so you've got very precise retention indices so with that retention index or an L-cane mixture you can start standardizing things and create a calibration file and that calibration file with amdisk and nist calculates retention indices and then you can start searching for those matches you can also start comparing against the blank come back just to make sure you're not identifying any false positives so if you're running amdisk you can create a calibration file this is what a calibration file will typically look like once it's run then you can calibrate when you call up the calibration file so that changes all the retention times to retention indices so now everything's kind of normalized and you can use the retention index information to help confirm an identification and then you can start searching the nist database using the software that they have so here you're seeing a peak and there might be in this case three or four possible compounds and then by clicking on the peaks the red peak, the yellow peak the blue peak, which is this curve then you can see what the mass spectra look like so this is for 11.597 minutes or retention index then you can see what's corresponding to the peak and you can see the location you can zero in so here we're looking at the peak which is marked in the big red box you look inside and you're seeing a yellow, red and blue some to the observed peak and then you can look at the masses and you can look at the match factor and you can see that under this peak we've got a good match factor, 84% or 840 and it matches to veiling there's maybe another peak which is weaker or another set of compounds and that might be something other so you can use the nist or amdus approach but there also is an automatic approach we'll talk to you guys and show you guys later called GC AutoFit instead of the amdus tools people have developed other tools commercial ones, analyzer pro from Spectralworks chromatops from LICO and these are compared amdus chromatops and analyzer pro four or five while six years ago now there's also other databases other than NIST 11, NIST 12 there's a GOM database which is in the Max Planck Institute in Germany or one of them and then there's the Oliver Fiend that's produced a semi-commercial library as well so a lot of the things, the processing techniques that are done for amdus and NIST can actually be automated so we've actually converted that to an automatic tool called GC AutoFit so it's a web-based tool like Bazel it still means you have to collect some reference actually you have to collect the sample you have to collect the blank and you have to collect the L-Cain standards but it does the reference retention index sort of fixing it does the peak identification that amdus does it does the peak integration which amdus doesn't do to get your calculation and concentrations it takes a bunch of different files it's quite fast, much faster than Bazel and it's able to identify quite a few compounds it doesn't work for everything but it works for mammalian blood, urine, saliva, and CSF but if you don't follow the protocol it won't work so that's another lesson or reminder so this is a quick outline of how you would run or prepare GCMS files you would have your L-Cain standards you can label them depending on what format they're in you'd have a blank file format and again you can label it whatever you wish and then you have a set of sample files so if you're analyzing a bunch of runs 10, 20, or 30 you can upload those you can do some conversions NetCDF and MZXML are the required ones they have tools that can do that conversion software that's either downloadable or associated with other instruments once you have those files there are the sample files they might be zipped files or you can upload them one at a time so you identify your L-Cain standards browse select blank files, browse select sample files, browse select and then you also need to choose your database so the database is a library that again is working with what you think you're analyzing so if you're going to be analyzing blood don't upload the cerebral spinal fluid library if you're going to be analyzing urine don't upload a CSF library so there's different types of libraries and there's certain types of standards that you can choose to help calibration, identification, quantification so here you've got your choice in this case we'll be using an internal library we won't be using your own because I don't think any of you brought your own library but it's a library that's internal to the server in this case we're choosing urine and in this case we're choosing our calibration standard of cholesterol which allows us to quantify things once you've uploaded things double check to see your alkanes standards see if they look nice look at your samples see if those look decent and then it crunches away and kicks out a list not unlike what you saw for Bazel which is a collection of peaks and retention indices what the compounds are and then it annotates them and you can view them using a viewer similar to what Bazel has you can also save the files on the CSV table and then of course you can view them as we've just seen on the spectral viewer so that's an automated form of GCMS and we'll run through that as well now it's important whether it's NMR, GCMS or LCMS just to be aware of the different levels of metabolite identification so in most cases what you're trying to strive for is what we call a level 4 or a level 3 identification so these things are positively identified so in that case it often means you have to have the authentic standard in your hands and compare it or in the case of NMR it's the NMR spectra because there's so many peaks match then you can be quite certain that that's the compound in the case of GCMS again if you have a high number of those matches to the fragment pattern and the retention time then you can probably put that into a level 4 but if you're just trying to match only the parent ion mass or a parent ion mass and a retention time then it's a little more tentative and it would be a level 3 that's where a lot of people do or leave things and I think this is an unfortunate situation that's going to lead to many problems so a lot of people identify compounds purely by mass matching and just to the parent ion that's a very weak identification and I would put it more at a level 2 and more often it's wrong than right so if you want to positively identify a compound we strongly suggest that you either identify it by a robust method like NMR or fragment ion matching by MS that's been coupled with retention indices or retention times or you use the authentic standard so level 1 is sort of the unknown which is a feature or an unknown peak and again that isn't terribly useful for writing up or thesis but in tabloid mix there are lots of unknowns so it's still worthwhile identifying them as unknowns so LCMS is very similar to GCMS if you remember the picture I showed for GCMS these are identical but conceptually it's the same thing if you want to be able to look at what's under an LC peak several compounds are typically there several spectrum maybe there they might not be as complex as the ones I'm showing here unless you're doing MS-MS but even with soft ionization you'll get some fragmentation so there's a number of commercial tools that people can use Agilent, Mass Hunter, Thermosev, Progenesis for Waters Profile Analysis for Brooker but there's some freeware that's available XCMS and MZ Miner the two that are most commonly used how many people have ever used XCMS? two okay how many have ever used MZ Miner? one okay, two so anyways how many have used commercial software for LCMS and tabloid mix? one, two, three, four, five okay so anyways neither here nor there, I just wanted to know what people know about so we're going to be learning about XCMS online and XCMS was a downloadable program it still is a downloadable program and it was one of the first open source tools for mass spectral processing so you can download XCMS you can run it and we used to offer a little lesson on how to do it it does peak picking, peak matching but now instead of a program, it's available as a server and servers are always easier to use than downloadable programs so we're going to use the XCMS online which is the server except it's a wide range of formats much more than the GC auto fit because it has to deal with all the different manufacturers and they haven't been able to agree on formats yet now in XCMS it doesn't do satellite identification or at least not in the way that we'd like it to but it is linked to a database called the Metlin database which you'll have an opportunity to use or explore so this is outlining how the XCMS or XCMS online works, it basically takes a large collection of LCMS data it'll get the extracted ion chromatograms and because retention times and retention indices in LC are not very uniform, not like GC they have to be aligned so they have to do things like what's called non linear alignment or time warping alignment so they'll shift and adjust peaks so everything matches once all those spectra which barely aligned initially are fully aligned then you can start basically running XCMS and that's when it starts identifying peaks, it starts separating them, starts measuring approximate amplitudes separates signal from noise and so on and it'll generate a whole bunch of M over Z measurements from there you can also if you run a tandem MS spectrum you can also pull those out from that and so from either the mass spectra if they're accurate masses or from the tandem mass spectra you can compare them to the Metlin database or the HMD database or other databases to identify your compounds in terms of peak detection it'll try and sort of combine peaks from the extracted chromatograms it'll do some filtering to make sure the peak is well detected so there's a fair bit of processing just like with NMR you can see the peaks are not always pretty but peak identification is critical this is an example of what the alignment corrections will look like so initially if you're running the same sample in this case maybe six or seven times you'll see that just from the drift on the column peaks will be coming out differently but in this case it was exactly the same sample so they should have come out exactly overlapped so what the XCMS software does along with any other commercial software tools is it will do this time warping or non-linear alignment so all the peaks will stop, it'll make the appropriate shifts in some cases it's just a simple linear addition, in other cases it's sort of a stretching out that actually occurs so before and then after so XCMS is compared to a bunch of different tools spec array, X-Line MS and spec, 10-Z-Line and in terms of this is published a number of years ago but overall it has or had this precision and recall all the other tools are obviously in their second and third iterations so they're doing quite well so they're all doing pretty well in terms of identification so if you want to use XCMS you can download it but today we're just going to be having people sign up and use the online version now unlike Bazel and GC AutoFit which are pretty fast and only run on a single CPU and XCMS is very compute intensive and runs on a very large compute farm so the scale of MS processing, data processing is much more difficult computationally than it is for NMR or GCMS this is just a very quick one I'm not going to go through this in detail because you guys are actually going to see this in the lab but it's just go to the website, create a job upload your data upload your spectra there's a couple of selection steps again you guys can refer to these slides to actually run it choose your parameters that are function as a type of platform that you're using, we'll tell you which one you're supposed to be using but if you're running mass spec they have pretty wide range of platforms that are supported then you submit and then it'll give you the status updates and report how far things are proceeding and whether it's ready to be viewed it's not instantaneous not one minute, not five minutes but I don't know how long for the samples today might be 20 minutes we'll see, we'll see if we can bring the whole system down once you've got the processing is complete then it has some wonderful spectra and views we'll try to explore things in a little more detail but what you're mostly interested in is your peak list table which in this case lists masses there are attention times, intensities which give you a relative measure of quantity not really a precise measure but that's that's the ultimate thing that you're hoping for so whether it's basal autofits fit or XCMS online all of them produce a list of metabolite names or identifiers in this case M over Z values and relative concentrations or absolute concentrations so in LCMS not all peaks are real so peak picking is never perfect the intensity is only relative and they are not concentration values but they still are useful for doing statistics peaks aren't identified with compound names so you still have to do some annotation after this so that can be a little challenging and that's where you use separate software or use tandem MS data to do some matching and comparisons so we've talked about how NMR is good for water soluble compounds, GCMS is good for organic acids and amino acids LCMS works well for lipids, for bases, for fatty acids generally better for hydrophobic molecules to get solid identification gophers to level 2 to level 3 or even level 4 you need retention time information you need MS MS data you need accurate MS data and ideally you need internal standards so although LCMS gives you lots and lots and lots of information and allows you to identify many potential compounds it's non-trivial to get absolute quantation identification so that's be warned it's appealing but not simple so how do you identify these compounds if you've got your masses accurate masses there are searches you can use a database called kebi chemicals of biological interest PubCAM which has 30 million compounds which only maybe 0.1% are actually natural products or metabolites so common mistake many people make is they just search through PubCAM because it's the biggest database well you're also going to get some pretty strange hits that are not at all biologically relevant but correspond to compounds that have never left the laboratory which is sort of the British equivalent of PubCAM very interesting database and then I mentioned the human metabolism database which is mostly restricted to human or mammalian metabolites but actually covers a fairly large swath of metabolome space even for plants because people eat plants and so plant metabolites end up in the human so here's PubCAM search you can search by molecular weight ranges and it'll give you hits and list these if we typed in this range from 89 to 89.099 we got 408 hits you can browse through to see if they seem realistic or reasonable kebi you can do a molecular weight search these are mostly biological molecules but they also include pollutants and drugs and a few other bizarre things and they cover mammals and plants and bacterial and fungal metabolites so there's a molecular weight search there and you can also do more sophisticated searches not just with molecular weight searches but actually with MS-MS or EIMS spectrum you're not just putting them in the single molecular weight you're putting in multiple peaks so these are the fragment ions so we've already talked about the NIST database I've mentioned the Metland database I'll mention a couple of other ones that are also useful so they do the trivial molecular weight search but they also support looking for positive and negative ion searches they can also search for the neutral mass they can do things like adept searches as well they can do MS-MS searching so these are much more useful as a rule for metabolite identification so one tool we've developed is a database that you can search and compare MS-MS spectra it's calculated or generated MS-MS spectra from all the compounds in the HMDV and all the compounds in the KIG the prediction tool is very accurate and so you can try and see if it matches any of those searches and that's one way of identifying by mass spectral or fragment matching you have three options you can have an option for it just take a compound and predict the MS-MS spectrum but another option where it will do the compound identification and for our purposes that's probably of the greatest interest if you've chosen that then you can enter the mass values the intensities it has an example that you can use and once you've entered that then you can enter at high energies medium and fragmentation energies are low fragmentation energies that's up to you it's a function of the type of mass spectrometer you're working with once you've submitted it then there's a comparison calibration and that puts the hips and you can see the spectral matches with the blue and red lines so as I mentioned with mass spectrometry you'll often get things that are fragments or adepts so you can get neutral loss species multiply charged species chloride addicts, sodium addicts potassium addicts in fact as you can see in an LC-MS spectrum are actually from these so called noise sources and it may even be higher than that so the challenge then is to try and get rid of these fragments distinguish the addicts, the multiply charged species as well as the isotopes for higher resolution mass spectrum and to group them together so this is an example we saw it with chlorine and GC-MS but this is with sodium and ES-MS so the actual parent mass of this compound is 951 dolpens but you'll see another peak above it which is 973 and so if you're just trying to do mass matching and you didn't know there was an addict you would enter 973.287 into METLIN or HMDB or PUPCAM and you'd get a hit but you'd be matching the wrong compound to be a mysotope or an addict and so addicts will form on negatively charged groups nitro groups, sulfate groups anything that has a strong negative charge so people have created tables all over Raphine and others of the types of addicts or masses that you'll see that you should generally look for and so by looking for pairs of peaks that are separated by 45 dolpens 17 dolpens, 28 dolpens 12 dolpens and so on you can identify things that are either addicts or singly charged or doubly charged or triply charged groups and so you can see from just a single compound it's possible to end up with many many different types of peaks and that's why there's so many of these sort of quote noise peaks that can be confounding and confusing but all over Raphine has a very nice list of addicts, a very comprehensive one as I said where the addicts is also the neutral loss fragments, these are spontaneous fragmentation events that will happen in EIMS and they'll create peaks so these are sort of fragmentation that you'll see if you've ever done MS, MS you've just simply done a direct injection but you're simply seeing these extra peaks and this is just because things break up so different tools databases, online servers metlin and ZDB hMDB, handle and predict addicts they're also able to predict dime pairs, a multiply charged piece metlin handles neutral loss species so if you're only searching by MS and you didn't worry about neutral losses and addicts, you're going to end up with lots and lots of mistakes, lots of false positives so the software that people use and it's partly embedded in XCMS online but a lot of other things is you want to remove and consolidate your addicts and your multiply charged species you want to remove and consolidate your fragments neutral losses, the breakdown products, any rearrangements you want to remove and identify isotope peaks called deisotoping then you want to remove any noise peaks from the sample blanks so this is why it's always important to run a blank or to check to see if the replicates the technical replicates don't show dilution so those are all indicators of just random noise that's showing up so these are all the things you have to do to clean up so if you had say a positive mode spectrum and you're getting like 15,000 features well if you remove the addicts it's down to 12,000 so if you remove the multiple charged species you're down to 10,000 if you remove the neutral losses you're down to 8,000 if you remove the isotopes you're down to 3,000 if you remove the noise you're down to 2,500 so you've lost factor 6 so the final spectrum might be 2,500 peaks that are real peaks and even then there's other issues that might not be that they're real compounds and then you'll do the negative ion mode and you'll get as many and so the tools that help you with that are things like MZMind there's a tool called Metfusion Magma, this is another one as well as commercial software we're not going to have time in these sessions for you to get familiar with these and to process them but again I want to press upon you that as rich as the LC-MS is it's a lot of work to get compounded notifications to sort the noise from the real peaks and it's not particularly quantitative because we're just talking about these relative peak intensities this is a quick question can you talk about the isotoping so when you get a mass spectrum of a compound you'll have its molecular ion and so on with its isotope so is it basically saying you don't have isotopes or is that very software dependent? what it's doing is really you're trying to take the five or six isotope peaks that are visible then you just want to consolidate them not treat them as six peaks but treat it as one peak but then the denoising issue if I got a very sensitive spectrometer and I'm not seeing any isotope peaks for this it's probably noise so that's another way of detecting the noise but again it's a function of how good and how sensitive the spectrometer and the mass resolution so in some cases the isotoping isn't necessary because you don't have the resolution so I know I'm kind of plowing through this we've got a lot of material here but we've covered a lot but we're just trying to highlight the different protocols used for identifying compounds one for NMR, one for GCMS and you'll notice I'm spending most of my time on LCMS in part because it is very complex so again the strengths of mass spec are that it's a lot of information but you can also with the best instruments highest resolution instruments get a lot of information to help identify compounds so if you can be absolutely confident that you've got the paradigm mass and you've been able to measure it to 0.1 ppm you're well on your way to identifying the compound so if you've got that high resolution of millidolms you can use molecular formula generators or you just put in the mass so six decimal places and a molecular formula so one example is a web tool called MZDB Maintained in Aberystwyth in Wales and they have tools for generating molecular formulas so you just type in your mass obviously your mass tolerance is usually much smaller than 0.2 dolms and it will generate the molecular formula if you've got your molecular formula then you can do searches through Pubcam so molecular formula is more useful than the mass because if you've got a formula then you can be there can only be one chlorine here there can only be six carbons it's not where you're trying to get you can rule out compounds that have fluorine or nitrogen or whatever so you can go a little further where if you look at not only the isotope peaks but also the abundance as well as rules about chemical bonding restrictions and presumed atomic composition data so generally if you're looking at mice and rats they don't take fluorinated drugs they don't take brominated compounds so you can exclude fluorine and bromine and other things from being in there so you can actually reduce the number of matches quite a bit and this is something that was developed by Oliver Fienden group called Seven Golden Rules for Formula Filting so you can take the accurate mass but if you use some of the other things like isotope intensities and consistency for bonds and valence, constructs, everything else you can narrow things down quite a bit both to the type of the formula and the possible structures and this is sort of shown here back in the early days when PubChem was much smaller but you can have 8 billion possible compounds with elemental compositions less than 2,000 Daltons and you can end up with from those 8 billion about 600 million compounds that are highly probable using the Seven Golden Rules and then if you look at PubChem's 10 million compounds at the time then it reduces it to much smaller and then if you exclude PubChem the synthetic compounds then you're basically left with about 50 to 100,000 natural compounds and so this highlighted also the utility of actually working with databases that were largely with natural products rather than say the PubChem database which covers anything that's ever been synthesized under the sun and the percentage of which never has left a lab therefore will never be in any organism. This is another plot that came from Oliver's description just showing that the large number of molecular formulas that you can come up with based on the masses and mass ranges and obviously as you increase the mass there's going to be more and more possible molecular formulas but what's also pointed out in the paper is that as you increase the molecular mass accuracy going from 10 ppm down to 0.1 ppm or if you improve your understanding of the isotope abundance you can very, very quickly narrow down the number of possible formulas from hundreds down to just one. So that's a very powerful filter and can be very useful in identifying compounds and this is just illustrating how mass plus the isotope abundance gave exactly the right formula for this compound. Now it doesn't give you the structure but it said given this very, very large molecular weight which could be potentially many, many things because there is accurate isotope abundance information and because the mass accuracy not great but still good enough was able to come up with this precise formula which matched the compound. So I think when you're searching through databases it's important to think of a few things. So the PubChem database as I said is kind of useless for metabolomics unless you can confirm the hit is a metabolite which then means having to check against a metabolite database which is sort of two steps. Kebby includes a lot of biological compounds but it's not restricted to any species. So it has metabolites from anything as long as it's alive. Metlin originally used to be a human metabolism database but it now is expanded into all kinds of plants and insects and so there's no origin. You don't know what these compounds are from. So a lot of people and I'm seeing papers where people submit or how, I ran Metlin, I got my hits and I was running on mice or rats and they get hits to all kinds of human drugs. And the mice and rats are not on human drugs. It's because they're not worrying about the origin of what the hits are. Same sort of thing if you're doing plant metabolomics and you're getting all kinds of human drugs that's not right. NIST has lots of pollutants and non biological compounds so those also have to be considered. So make sure you think about the organism and the source and if you can use organism-specific databases generally your hits will be much more robust and accurate. So seeing that NMR is quantitative GCMS can be quantitative. MS generally is not. Most studies are not absolutely quantitative. They will give relative measures. To get absolute quantitation you actually need isotopic standards. Caterium, carbon or nitrogen 15 labeled standards. And you also have to have standards that are either the same or very similar to the target chemical that you're working with. So to do that you use what's called single reaction monitoring or multiple reaction monitoring. SRM or MRM. Is anyone ever done SRM or MRM? 3, 4, 5, 6 So that allows you to get quantification and again it's using a compound that's appropriately labeled. It's selecting for that compound or its fragments and when you have those fragments you can then compare peaks and peak intensities and because you know exactly how much you've added in terms of that isotopically labeled standard you can get precise quantitation by measuring the area under the curve. So it's something that you can do manually and it's a fair bit of work or there are now automatic or semi-automatic ways of doing that. And so one of the best ones is a kit produced by a company called Biocrities where they have created and have all of these isotopic standards and allows you to measure anywhere from 160 to 180 different compounds and get the absolute quantitation. And they have other kits for measuring bile acids and I think there's another one coming out for steroids and there's a trend now in many other organizations to start creating kits to do quantitation via mass spec and if you ran a kit say on your end this is what you typically would see with concentration of metabolites and compounds identified and you can see with the remarkable sensitivity you get with MS getting concentration ranges from 10 nanomolar to 7 millimolar so a 10 to the 6 full difference in concentration. So if you do things right with mass spectrometry with LCMS you can get levels of quantitation and identification that are better than GCMS and they're better than NMR but it's a lot of work and if you don't do it right you can get some pretty bad results and I just want to emphasize that there's too many MS based metabolomic studies that are appearing that we're just not being carefully done so don't be lax careless with analyzing MS MS data okay I think we're about due for lunch actually


The leading system for an exchange capacity, the Central Bank of Nigeria's left team of its ban on sourcing of forest from official market for the importation of 43 items, please the business community that raises some questions. The Apex Bank announced that it was raising dollars of life to the market and freeing import is to obtain forest from the official window. While its advocates have strong arguments justifying the unbending critics, however, some items on the list, the decision of the Central Bank to leave the ban on 40 trees, item signifies a major step in resolving Nigeria's forex crisis, as most critics have called on the Syrian on the eminently to lift it. However, some of the items remain on the list, the ban and customs list rather, and it is unclear if they will be funded for forex purchases. It is also unclear how much demand this will drive to the official I&E window, and if the Central Bank has the capacity to meet this demand in the short term until liquidity returns as it is claimed. On the show today, we'll keep our eye on a CBN's willing buyer, willing seller's principle. Welcome to Business Insights and Plus TV Africa, I am Justin Acadone. Just before we get into the discussion proper, my dream government and businesses have an edge to keep an eye on the rise and wave of a new variant of populism among the youth as it could upend both government and businesses. This call was made during the public presentation of the distillation edition of the periodic report of a public relations and communication consulting firms in Lagos. Details in this report. It is a public presentation of the distillation edition of a periodic report that is known as the neuroscience of Nigerian youth, trend and culture report, fittingly-themed, a reminder. It is about the rising wave of a new variant of populism amongst the youth. According to the organizers, the rise of this new variant is challenging for governments and businesses all over the world. Chief strategist, chain reaction Africa, Israel O'Kane, believes that the businesses and the present administration must make diligent efforts to study and understand the trend. Why are we tracking trends? Why are we tracking culture? There is a saying that culture will eat strategy for breakfast. No matter how intelligent the people in government are, unless they understand the emerging and the external cultures within their own jurisdiction, then their policies are bound to fail. O'Kane explained that the mystics surrounding behavioural evolution within the youth's population can be perplexing unless intentionally stoddied and understood by those responsible for policy and business decision-making in government and the side of businesses. Of the trends we've discussed in there today has been the Afrivas, which has to do with the rising sentiment, the rising consciousness, the Afrocentric sentiment amongst Nigerian youth. And examples were given that even government, even in naming your policies, even in naming your programs, name them in such a way that the Nigerian youth can hone them, can feel that this belongs to us, not giving policies for the names. A commissioner for information and strategy being a immortal show and his counterpart from the Ministry of Youth and Social Development, Mbolaji Ogunlede, who represented the illegal state governor and his deputy, affirmed the importance of the youth's population in the society. Our rising government is a very big lesson that we need to find a way to... the kind of language that we used to talk to our young people. We need to find ourselves in their space so that whatever we need to do for us to improve as a government, as a community of people who understand what the future means to young people, that we need to do it. It's all about engagement. Engagement in the sense that when the youth come out to protest or rise to an occasion, they are fighting for justice, they were fighting for our voices to be heard. So we just need to ensure that the people who are supposed to hear are hearing and they are acting on it, they have to be proactive instead of reactive. They however cautioned the Gen Z, not to jetters in the old, in the criss, for metaverse and preoccupation with the realm of alternate reality. My advice is that our young people should try to have a marriage of the old and the new, because in the old you find wisdom, in the old you find self-regulation, apart from what's... as different from what people see now as a license for attitude of, I can do whatever I like. The distillation addition of the report focused on five other key trends, namely afri-verse, which captures the rise of afro-centric consciousness amongst Nigerian youth and Igubeta, among others. Love Ikuku Uyiduku, plus TV news.


when the 2020 season begins, there will be some rule changes. These are the most notable ones reportedly upcoming a 26 man roster expanding to 28 in September. A three batter minimum for pitchers excluding inning ending appearances and then the IL changing from 10 days to 15 days only for pitchers not position players. So let's begin with the three batter rule. It has been a point of contention. You want that lefty, lefty matchup. But guess what, Flash?  But I think if you think about the Yankee bullpen, this rule is probably not going to bother them more than other bullpens in the league because to your point, we have Zach Britton who can get lefties or righties out. We have out of you know, a right hander can get lefties or righties out. Chat Green doesn't matter lefty or righty. So I think they're better equipped to handle this. I think you're gonna get in situations late in games where maybe you have a four run lead and you wanna throw one of your lower leverage guys out there, he's gotta face three batters before one of the big boys comes in after them. But all five of these guys, doesn't matter lefty, righty, Jack. They can get the job done, they can clean out an inning. Now you look at the splits they had from last year and you're absolutely right. The only guy who had a little bit of a different split out of being a 177 against right handed batters, 241 against lefties. Still a low batting average against lefties. But I wonder about Aaron Boone's strategy and how he's going to handle this going forward. He is not probably going to be too impacted by it, but I do think there will be some situations where as a manager, you were ready to reach for that phone after one batter. There were times last year where they would bring an autovino to face one guy and then would go to somebody like Britain. Well now you have to sustain having that pitcher in there for three batters. How does that impact you? I don't love it. I would rather the manager have the opportunity to decide which pitcher he wants to face that batter at that moment. I don't love the fact that Major League Baseball is dictating you have to take a three batter chunk. And they did this to try to speed the games up, right? So my question is how much is it really going to speed up the game? A couple of minutes at the end of the night, that's one of the things we'll obviously be paying attention to during the year. We talked about Aaron Boone a lot in this segment and I'm anxious or eager to talk to him in spring training and get inside of his mind a little bit. He's going to have to, I believe, talk to some of his relievers and let them know these guys know the rules. They're going to know that the scenarios are going to be different, but I think they're all going to have to be a little more adaptable going forward. What about a new pitching coach coming into the equation? Larry Rothschild, that veteran guy, the veteran voice that would be in Aaron Boone's ear, he's not going to have that this year. No, and we're going to have to see how that works out with Blake. Blake getting his first opportunity to coach at the Major League level. The Yankees are very high on him. They love his mind as a pitching coach, but Blake has already even done interviews where he has said, I've never sat in a Major League dugout and had to be the guy who makes that split second decision or refers to the manager. Hey, this is what I think we should do here. So definitely a lot to focus on with these new rules.