Welcome to MOOC course on Introduction to Proteogenomics. In the first module you learnt about genomics. In the second module we started discussing about proteomic tools and in this slide in the last 3 lectures I try to give you the basics and foundation of proteomics. Today lecture is going to be conducted by Dr. Karl Klauser who is a principal scientist at the proteomics platform at the Broad Institute of MIT and Harvard. Dr. Klauser will focus on the basics of mass spectrometry based proteomics with emphasis on the electro spray ionization, the factors which influence good ionization efficiency and the architecture of a mass spectrometer. So let us welcome Dr. Karl Klauser for his lecture. If you have seen the description of what this session was supposed to cover it looks like this. As I put all the slides together I decided not to have them in quite that order so the general flow of sections that I am going to go through is illustrated here. And so let us get started. All right so the first section I am going to teach you some of the basics of mass spectrometry. Today in our lab in Boston we do a range of types of experiments in proteomics including discovery based experiments and targeted experiments. These are examples of all the different kinds of instruments that are available from the manufacturer of thermo. We have at least one of each in our lab and all of them have to do some of the same basic functions. They have to create ions from peptides separate based on charge and mass. Although we do mass spectrometry we do not actually measure mass we always measure mass to charge ratio. The instruments basically do two types of generate two types of spectra. An MS spectrum or an MS MS. The MS spectrum measures the complete mass of a peptide and the MS MS spectrum is after there is fragmentation and you measure the masses of the fragments that are produced from that spectrum or from that peptide. All right the as of today in proteomics there is essentially one major ionization technique that dominates the field and that is electrospray ionization. If this was 10 or 15 years ago there would have also been some Maldi or matrix assisted laser distortion ionization but today most of what we do is really driven by electrospray. So, this is a depiction of what is on the outside of the mass spectrometer here at atmospheric pressure. You end up with a liquid that flows out the end of an LC column. There is a voltage applied that causes droplets to be formed that exist as ions and then those ions are transmitted into the mass spectrometer and along the way they have to become desolvated. This is a cartoon representation this is an actual picture of what that electrospray looks like and the liquid that is flowing is most when doing proteomics most often it is through a liquid chromatography interface flowing at the most common scale to do this is 200 naniliters a minute can't be flowing salt or detergent peptides have to be in water acetonitrile and dilute acid most often that is formic acid about pH 3. If you have salt or detergent still in your sample you end up gunking up the front of the mass spectrometer and it doesn't work as well. We measure mass. What is mass? So, the elements that contribute to proteins are dominated by hydrogen, carbon, nitrogen and oxygen. The masses are shown here. It is important to understand that each of those atoms or elements does not exist as a single species. They have an isotope form. Those isotopes are not very abundant. So, you can see the one with the greatest abundance is carbon. And so 1 percent of carbon exists in the C13 form. We are also going to be able to take advantage of that. It is crucial for doing quantitation these days for there to be the existence of carbon 13 and nitrogen 15. So, one other aspect that I want you to keep in mind is that the difference between carbon 12 and 13 is not the same as the difference between nitrogen 14 and nitrogen 15. That is going to be very valuable to us when we talk about multiplexing later. So, it was actually I have been doing this for about 25 years and it was only a few years ago that I was looking at something like this and I said wait a minute a neutron is has a different mass depending upon whether it is attached to nitrogen or to a carbon. And the answer is yes of course because we do not measure the actual mass of something. We measure the mass based on energy. And the binding energy of the protons and the neutrons in a nucleus is different depending upon which element you are talking about. And that is the source of the difference between adding a neutron to a carbon or adding a neutron to a nitrogen. So, once you get some data out of a mass spectrometer these are some of the basic characteristics of it. I told you that there was isotopes. So, instead of getting a single peak we get multiple. This first one the leftmost peak or lowest mass peak is referred to the monoisotopic peak. That means it was measured of a molecule that contained entirely the first isotope of the elements that it contributed to. Because carbon is the most abundant isotope the major contributor to the other isotope peaks is carbon 13. So, here you have one carbon 13, two carbon 13s, three carbons, four carbons. But I told you it is only 1 percent. This is huge it is almost as big. Why? Well, once you add up something that has a intact mass of about 2000 there is going to be hundreds of carbons. So, the chance now of being at least one of them being carbon 13 goes way up. So, once you have these isotope patterns there is also some extra valuable information encoded in them. Approximately the mass of a neutron is 1, but we do not measure mass we measure mass to charge ratio. So, it is m divided by z. In this case you can see that those isotope spacing is 0.25. That means the charge now is 4 and we can determine that because of that isotope spacing. On a high resolution instrument this is what an isotope cluster will look like. The numeric value assigned to resolution is something like 60,000. That is the typical resolution that one will run an MS-1 scan on when using an Orbitrap instrument. Where does the 60,000 come from? The typical measure of resolution is m over delta m. So, that means you would use an enumerator you would use 431, but the delta m part comes from the width of the one of the isotope peaks. So, that is very narrow. When you have a low resolution instrument 2000 would be the resolving power of this. Now it is also kind of complicated when you compare one instrument from one manufacturer to one instrument from another manufacturer because although they both use them they will all use the measure of resolution. The resolution is not uniform across the mass range of an instrument. And the particular manufacturers do not necessarily quote their resolution at the same mass. And furthermore to make it even more complicated thermo uses different measure depending on which instrument they are talking about. So, they will quote you a resolution number at mass 400 for their quadrupole type instruments and for the hybrid instruments they will quote it at mass 200. So, when you start to say oh I want this instrument because it has got higher resolution it does not. It is just they have changed where they give it to you. But what you really need to know is how narrow are those peaks going to be and are they narrow enough to do things that are useful. TMT-10 resolution is something we are going to talk about later. And the ability to resolve the N and C isotopes of TMT report rions is important. So, I have already said the gap there was 0.25. We measure amino acids. Kelly showed you earlier when you are working with DNA or RNA there is only four things. And they are not glycine 3 and aniline and cysteine which is what they would be if they were talking about amino acids. There are there are 20 amino acids they are depicted here. This organization of the amino acids has them showing different properties of the amino acids. And I was told that there might be a quiz conducted throughout the course of the workshop here. And it just might be useful to know that my personal favorite amino acid is probably. The mass of tyrosine is 163. A couple slides from now you are going to need to know that. These particular amino acids over here, leucine you cannot tell the difference between leucine and isoleucine with a mass spectrometry because they have the exact same elemental composition. But from the standpoint of measuring ions these ones are kind of boring. They do not do much. The reason proline is my favorite is because it causes all kinds of havoc. The side chain is bound to the backbone and it likes to put retain charge there. And so you get a nice huge peak in an MSM spectrum when there is a proline and you get an ion cleaving on the N terminal side. We could not do proteomics if it wasn't for these three amino acids in particular lysine and arginine. They are basic. They bear charge. They are the basis for making the ions have charge. We do positive ion mass spectrometry. It is possible to do negative ion mass spectrometry. I have been doing this for 25 years. I never do negative ion. To me if it was negative ion it would be totally boring because it cannot do peptides very well that way. But I give you some property information here. The pK of the basic group on the side chain is quite different and the arginine has much more basic. It really wants to hold on to that charge much more strongly than do lysine or histidine. Do you have a question or a thought? Please feel free to interrupt me. So later when we interpret spectra by hand, if you knew all the masses you would be able to keep up with me.  So after we do some math I can just tell you right away what the amino acid is. You are at least going to know tyrosine is 163. Okay. Here is an old slide that illustrates an MS1 spectrum. There is a few things I want to point out here. This is measuring multiple peptides at one time. And you can see that this one is a singly charged peptide. Its isotope spacing is 1.0. This one over here is doubly charged. And then I think one is triply charged here right. So the spacing is 0.3. You can tell for the resolution or the peak widths there that this is an old instrument. Okay. Those things are wide. Okay. If we do label it a little differently, one of the things you can see is that the masses of those peptides after you account for the charge convert it back to singly charged. This peptide has no basic residue which is why its easily singly charged. This peptide can hold two charges. And you can, you typically will have charge on arginine and lysine and then on the interminous as well. Okay. It is also possible to hold charge on asparagines and glutamines. Okay. But here we have easily two basic residues. When they are right next to each other it is hard for them both to be charged. Okay. But here this one has got enough basic residues. Peptide 3 is triply charged. If the mass range was expanded wider some of these peptides would produce multiple charge states. Okay. So you would see 2 and 3, 3 and 4, something like that. All right. So how do we do MS and MS-MS? Okay. We use one instrument. Okay. But it is going to do two things. And this is the simplest instrument where there is one mass analyzer and a second mass analyzer with a collision cell in between. Graphically this is equivalent to a triple quad instrument where to do an MS measurement you let the first quad just pass everything through and then we turn off the collision cell and we measure every ion that went into the instrument and that gives you intact precursor ion information. You may hear me switch between the terms precursor ion and paranion. Okay. When I am doing that, when I say paranion I usually mean the singly charged version of the ion and when I say precursor I mean the multiply charge that is not universal. It is just my personal way of distinguishing what I am talking about. All right. To do an MS-MS spectrum we set the first analyzer to only pass ions of a particular mass. Okay. And then the width that we allow might be only about two daltons. Okay. So only things that have, okay. And in this figure it is about, it is an old figure that width was 4. Okay. So we are going to allow anything with a mass of 834 to 838 to pass through. Collision cell is going to turn on fragments into pieces and you get an MS-MS spectrum. Okay. You could put three mass spectrometers together and you would get MS to the third. Okay. That works a little bit differently, but the principle is the same. Okay. All right. The mass spectrum right now is labeled b and y and we will skip ahead to talk about that. All right. After this slide we are almost never going to see elements again. We are always going to talk in terms of amino acids and we will use letter codes for amino acids. But I want you to keep in mind that a peptide is actually a molecule. Okay. And it has bonds between carbons and nitrogens and oxygens. This is a peptide backbone and if you look close you will see that there are essentially three different types of bonds here. The most common one that will fragment in during MS-MS is this one. Okay. And that will lead to a b ion if the charge is retained on this side of the peptide and it will lead to a y ion if the charge is on that side of the peptide. Okay. When we have a mass spectrum it is not the measurement of one ion. It is the measurement of many ions. Some of them are going to fragment here. Others are going to fragment there. Others are going to fragment different places. And so the spectrum is the sum of all of those events being measured together. Okay. Why would you call this b and y? All right. Well, that is because there was three things. Okay. So we call them a, b and c or x, y and z. The most common dominating ion types though are b and y. Okay. But it is not quite that simple. You can also lose break bonds that are in the side chains of the amino acids. Okay. And in particular you can lose water and ammonia dependent upon having these particular amino acids in those side chains. If you have a phosphorylated residue you can lose 98 from the side chain of serine and threonine. Okay. Again, you can also have, if you start out with a triply charge or doubly charged peptide, the fragments can have multiple charges on them. Okay. All right. This is the way a spectrum comes out of the instrument. It's, there's no red, there's no blue. It's just a bar chart, black, numbers on it, and you got to interpret it. Okay. So this one by the way is a beautiful spectrum. Life would be grand if everything was like that. The pickets represent the peaks and the spacing would be nice and desirable and it would be ideal. Okay. If you had the choice to have a very nice looking spectrum it would look like this. Okay. All right. Now, what information content is present in this? Okay. There's a, so first of all you're going, whenever I show you these things there are several things you should know. This is the file name which means nothing to you but it tells me what project the spectrum came from. This little red carrot is the position of the precursor mass in the MSM spectrum. Over here is the parent mass which is singly charged. Here's the precursor mass m over z and then the precursor charge state there is listed as z. Okay. So if you look at this spectrum you can see there is spacing of peaks that looks pleasant. Okay. There's also some apparent symmetry to the left and to the right. Okay. That is, it is helpful to look at that symmetry. Okay. I have colored those blue and red because they're going to turn out to be b ions and y ions. We don't yet know which one they are but a b ion plus a y ion that fragments at the same place adds up to the precursor mass and that's the basis of the symmetry. Okay. So you, once you have two ions that are on the opposite side of the precursor mass symmetric they cannot be the same ion type. They must be complementary. Okay. So if you then start to do math or subtraction to figure out the mass gaps between them you don't need to do math on ones that are symmetric to each other. Okay. All right. So then we look at some of the mass differences from peaks and if you had memorized all of them you would immediately recognize that those are masses that correspond to amino acids. Okay. All right. Mass 163 corresponds to, come on, say tyrosine. All right. Tyrosine. So we can tell from this set of mass gaps that this, the sequence that's part of this peptide is going to be tyrosine, tyrosine, leucine or isoleucine. Can't tell the difference. Allanine, threonine, 115 is aspartic acid. Okay. And then this is a symmetric spectrum and you can find those same masses going the other way. Okay. I have already colored them blue and red but there is no obvious way to say that one is a b set and one is a y set. Okay. And so how do you, how do you decide? Okay. Well if you can get to the end that helps you. Okay. If you can get to, and in this case we can't readily get to the end in either case. Okay. This 201 there's going to be probably two amino acids that you have to add together to get to that one that's probably, that could be a b2 ion. Okay. This is, this comes from an older instrument where we don't have low mass available to us. Okay. This is a tryptic peptide. Okay. So that means it's very likely to have a C-terminal amino acid that is arginine or lysine. Okay. The y1 mass for arginine is 175. The y1 mass for lysine is 147. Okay. We can't, we can't readily get from 201 to either 147 or 175 in a distance that is consistent with a amino acid mass. So that suggests that that's more likely to be a b ion than it is a y ion. Okay. We could also figure out if we could get from here to the top which is, that's a 1364. Is that right? Okay. All right. So what I'm trying to show you here is that there's a lot of information. Okay. If we have a complete interpretation of the sequence it would be shown here. Okay. The way I have slashes between the amino acids is to indicate that there's fragmentation. This red slash indicates that there was only a y ion there. Okay. This says that there was only a b ion in that position and pink means that there's both b and y. Okay. So this peptide is fragmented nearly completely but it has not given us information about the order of the first two residues. Okay. Together they add up to 201 but we don't know whether it's AE or EA. Okay. And similarly over here we don't know the order of cysteine or alanine. Okay. All right. But as with mass vector or driving down the street in India not all two wheelers are oil end fields. Okay. Sadly. All right. So some you get peptides that will fall apart. Do not give anywhere near as complete sequence information. And it's actually much easier to manually interpret these things because you can't get very far. Okay. So the best we can do is we can tell that there's some symmetry there. We have at least one mass gap that is consistent with an amino acid. And then there are a couple of combinations of amino acids that will add up to 202. Okay. When you search a database this is the only peptide that will be consistent with all of that information. Okay. All right. So what I tried to illustrate for you is that unlike DNA sequencing where it's typically the case that you get a clear, complete answer at every step of the process, with an MSM spectrum you have incomplete information. Okay. And what I've tried to go through and tell you is some of the factors that contribute to that information being incomplete. Okay. And here's a list of those things here. Okay. The fragment ion types and the tendency of for them to occur depends a little bit on what kind of fragmentation mode you're using. There are different ones out there and I will come to that in a later slide. Okay. You have complimentary information in both directions. Okay. Which, but that can lead to some uncertainty as to which direction you're in. Okay. And I told you there were 20 amino acids and if you were looking for mass gaps you would hope to find one of those 20. But there may be other modifications that happen that you have to be aware of and that's going to alter the mass. Okay. Then there are some amino acids like I told you a leucine, niolucine where you can't tell the difference because they have the same mass. There happened to also be some combinations of things that have the same mass. Okay. All right. And or very similar masses. Okay. So lysine and glutamine for example both have 128 and if you have low resolution low accuracy you can't tell the difference. But today with an orbitrap type instrument you do have the mass accuracy to tell the difference between those things. Okay. Two glycines together is identical to one as faraging. Okay. A glycine and alanine is identical to a glutamine. So these things go together to make your life a little bit different, difficult. This is a figure that I made in graduate school because I was faced at that time with many spectra that were more like the poor quality one. Okay. And I wanted to know that this was before genomes were done. Okay. And I wanted to know once the genome is done are those poor quality spectra still going to be good enough to determine a sequence with a database of a complete proteome. The good news is I'm still standing here today doing mass spectrometry. So yes the answer is it's good enough. Okay. But this is the nature of the figure that I made. At that time the we were still unknown how many genes were in the human genome and one of the highest estimates was that it was 100,000 genes. Okay. So I used that as an upper limit and then I took the mean length of a gene or of a protein in the Swiss Pro database at the time and that was 350. And then I made these calculations. Given the peptide length here I wrote a program one of my first programs to go through and count how many peptides there were going to be at that length given these assumptions. Okay. And so the red line is the simplest one which is to say if you could make all possible sequences and count them this is what that's how many there would be. Okay. Then the blue line is how many peptides there would be in the human genome. Okay. And then the green once I found out that this was nowhere near close to the red line. I said well how little information could you actually do this with. Okay. All right. And that's where I calculated the green line. So the green line means we don't actually know the order of any of the amino acids. All we know is the amino acids that are present in the peptide the amino acid composition. And to my surprise at the time you could start to get out to a point where if you had long enough length the amino acid composition was going to be unique. Okay. So you can still do a lot of good with these partial sequences. Okay. Life of course is a lot easier when you get complete sequences but life is not always giving us that. Okay. All right. So programs then have been written to do this kind of thing and I've written one of them. The software package that I'm responsible for developing is something called Spectrum Mill. All right. And but nonetheless all of the software packages out out there do this basic type of thing. Okay. You'll start with an experimental spectrum. You start with a database of sequences. Okay. You have to take those sequences and digest them into peptides theoretically. And then you make a model spectrum from the sequence and you match those two up. If you get a match however you score it then what you're looking for is to find the peptide in the database that gives you the best match. Okay. All right. So when you do this if the database is incomplete then you can get the right answer. Okay. And that actually causes some trouble because the programs are always going to give you the best answer. And what you'd really like to know is can you only give me the right answer? And if it's not there could you please just tell me that. Okay. Okay. But they don't do that. Okay. All right. So that can be frustrating. Okay. All right. So as a consequence though now what we do is since we can't be sure we've got the right answer let's at least estimate what our error rate is. And so I'm going to talk to you about calculating a false discovery rate from this type of thing. So building further from where I started about the basics of proteomics workflows and focus on the mass spectrometry Dr. Carl has further given you the detailed concepts of the principle of electro spray ionization and why it is important in proteomics. Then he talked about importance of high resolution of isotopes for proper identification of peptide sequences. How collision induced ionization results in the formation of different types of ions which is useful for the identification was also covered. Then he showed you examples of different spectra and how those could be interpreted manually something similar to what I talked in the in the previous lecture. But now you have seen much more detail about how to derive the spectride spectra manually. Additionally you are also informed about the factors which could affect the fragmentation of peptide. And lastly an effort was also made to help you understand the architecture of two very advanced mass spectrometers used in proteomics. So I hope from my previous three lectures and Dr. Carl Clauser's today's lecture now you got a very good understanding about how to use mass spectrometry and different you know important considerations for doing proteomics using these kind of instruments. In the next lecture Dr. Clauser will help you appreciate the importance of sample preparation. And then moving on to the quantitative proteomics with the use of i-track and TMT labels for quantitative proteomic analysis. Further he will talk to you about use of various search engines in protein identification. Thank you.


Welcome to the Introduction to Astronomy. In this lecture we are going to talk about the formation of stars, so how stars go about forming from the material in the interstellar medium. Now let's just take a moment to review and go over some of the basics of stars. And we have stars that are, most stars are main sequence stars, much like our Sun, and they produce energy through nuclear fusion. That means they're converting hydrogen into helium at the rate of 600 million tons every single second. Now how much of this would depend on the mass of the star, a more massive star would be converting more, a less massive star would be less than this. Mass ranges of stars come from a little less than a tenth of the mass of the Sun to about a hundred solar masses. These small mass stars are more common, the main but the massive main sequence stars are hotter and brighter. The lowest massed main sequence stars are the ones that are cool and faint. And a galaxy like our Milky Way has the gas and dust to make billions of stars. So let's look at an example of one of these star-forming regions and that would be the Orion Molecular Cloud. This is one of the nearest star-forming regions, only about 1500 light-years away. Only a little bit of it is visible, much of it is very dusty and invisible to us. And we see it here, the four stars that outline the body of Orion are present as well as the three stars in the belt, which make Orion relatively prominent and easy to see. Down below we have the stars in the belt are about 5 million years old. So they're still relatively young. The sword hanging down from the belt contains stars that are less than a million years old. And the trapezium stars in the Orion Nebula here are about 300,000 years old. And we can zoom in, let's take a look at those trapezium stars. And here they are in visible light on the left, visible light on the left here, and in infrared on the right. And there we see those stars. They're hard to see in visible light because of all of the dust around them. However, when we look at them in infrared, the infrared penetrates the dust and gives us a much clearer view, not only of those stars, but of other stars, some of the 2,000 stars that are forming in this region. So this is one of those nearby star-forming regions. We see far more, in fact, far larger ones elsewhere in the universe. Now how do we go about clearing out the Nebula when the star cluster forms? Well, the stars will have strong stellar winds and that will clear away the material. And that is much like the sun clears out the solar system. It has a relatively strong stellar wind and that was able to push away at material. And we can see in our image here that when a cluster of stars is forming, the winds from that will push material back until it starts to recede. So it'll start pushing this material back outward and will clear out this. Now that will compress and we'll have more stars forming. So that will push away the remaining material. Massive stars do not live very long and end their lives in supernova explosions, which will also help push material back. Both of these compress the nearby clouds and continue that star formation process. So what stars have already formed are beginning to do the next set of stages. Now when we look at stellar birth and we see some areas of this, first of all, we cannot see it directly in visible light. Visible light does not penetrate, so we use infrared or radio waves. Infrared for the later stages and radio for the very earliest stages that penetrate the dust. The initial collapse is a very short period of time. Now here we see sometimes what is known as the pillars of creation, young stars that are in the process of forming. Now as I mentioned, you cannot see them in the visible light, but if we look in the infrared with the same area, and this is an image from the James Webb Space Telescope, we see how different they look, how much material is not visible. So we see some dusty areas when we look in visible light, when we look in a little more in the infrared, we see far more detail there and we see details in the structures. The places that are still dense are those dense knots where stars are forming currently. So you form a dense core within a clumps of material and most of that star formation is going on at the very peaks here, where parts are sticking up.  Now here we can see how it looks, what we feel stellar birth is like. We start off with a large cloud and we form a dense core within that material. So we start off by forming a dense core of material and then that begins to collapse down into a disc so it collapse along the axis and the gravitational force becomes stronger and becomes dominant and a rapid collapse then begins and material, it's much easier for the material to fall inward from the polar axis than it is along the rotation. So it's a lot easier to fall down and that's why things come into a disc of material. Now as that disc forms, the star becomes what we call a T-tory star, which occurs for stars that are similar to our sun. Again, it's hidden in the dust cloud so we need to look at these in the infrared. We also get very strong stellar winds that will come here and will clear out the remaining material. So here we see jets of material coming out along that polar axis so as the star is clearing out it actually sends out jets of material. It's hard for that material to come out along this axis. It's much easier to go out perpendicular to the disc that is formed and that will clear out the remaining material giving us a disc, the jets temporarily which form what we call a herbig aro object and we can see an example of sketch of one of those here. So in that we have the star that is in the process of formation. We have an accretion disc of material around it. We see the jet. The herbig aro object is actually where that jet of material strikes the interstellar medium and causes it to glow. So it's not directly the star, it's actually the interstellar, material in the interstellar medium being excited. So this is a sketch of how it happens. Let's look at an actual one here. So this is the herbig aro object. The star is hidden at the center here and the bright spots would be what we would see that would glow in brightness. That's where the jets of material strike the interstellar medium and begin to glow. Now these are the very earlier stages. Then the star starts to settle down. So the winds will diminish, the jets will diminish and eventually disappear. That disc will likely begin to form planets. So we'll start to form planets and we'll look at exoplanet formation shortly and the wind and radiation pressure clear out the nebula, emptying it and the star and planetary system will remain. Now we can look at this on the HR diagram which as you recall is a plot of temperature and luminosity. We look at the temperatures on one axis, increasing to the left, we have the luminosities increasing upward. And when we plot those, we find that most stars will live their lives on the main sequence here, which is the red line. But as a star evolves, it will change its position and what that means is that its temperature is changing and its luminosity is changing. Overall, the temperature will increase while the luminosity decreases. So that's what we see with any of these stars as they're forming, the temperature is increasing means they're moving to the left, the luminosity is decreasing meaning they're moving down. So the stars tend to move down and to the left. The core reaches a critical temperature when nuclear reactions begin and that is when the star settles in on what is called the zero age main sequence. That means that it's as it's when it first becomes a star. However, if we have an object less than 0.075 solar masses, the nuclear reactions will never begin. It will never reach a high enough core temperature for nuclear reactions. And that becomes what is known as a brown dwarf, which we will look at again elsewhere. So let's go ahead and finish up here with our summary. And what we've looked at this time is that star formation begins in a cool molecular cloud in space. The material collapses and we form things like disc and jets of material around the star and we can use the HR diagram to be able to study these different stages of stellar evolution. So that concludes this lecture on formation of stars. We'll be back again next time for another topic in astronomy. So until then, have a great day everyone and I will see you in class.


Oh, what's up everybody once again is brand man Sean and today we're gonna talk about sales funnels That's part two of my video talking about marketing funnels Now this marketing funnel and sales funnel content will be some of the most important content on this channel when it comes to you Building an effective strategy for whatever you want to sell, but of course often we're talking about Music and the merch and all them other artists related things So you really want to show this and share this with your team when y'all are trying to build out the strategy But anyway, let's hop into it. All right, of course in that first video I showed you guys the marketing funnel and as I said here is a sales funnel. There's some slight differences I'm just gonna knock out sales funnels and then we're gonna run into the nuances later So just follow sales funnels. You have lead generation lead qualifications selling and then CRM You'll find multiple versions of a sales funnel across online But I think this is the simplest one to start off with right so lead generation means you are Finding people who could possibly be customers. You're trying to start identifying potential customers Lead qualification means out of those people that you got in contact with that you made aware of you You're trying to qualify Which one of these folks are actually the type of people you want to sell to or could sell to So let me backpedal a little bit when we talk lead generation. There's two types outbound and inbound activity I'll get into the inbound later, but let's pretend that we're making a sales call You know those people who you have no idea who they are they hit you up on the phone those cold calls Well, that's somebody doing outbound lead generation. They're going out putting effort in To finding customers and then throughout those conversations typically they're trying to qualify These people so when we talk about qualification and seeing if you're able to be sold to Every single product isn't for every single person. Let's just go to the extreme and say I'm selling something. That's for senior citizens and I'm call you up I have no idea how old you are and I'm asking you questions on the phone to see if you're over 65 years old or whatever the senior citizen age is if I ask questions and find out you're under the senior citizen age Then I now know you are not qualified to buy Whatever I am trying to sell or maybe I might try to finesse the situation and see if you have a grandma or grandpa That I could get in contact with but that's how lead qualification works And then of course selling that's the actual sell in exchange of the product that you have customer Relationship management now in this phase you manage your relationship of The customers that you now have you and you can you can also keep it going with just a quality product experience Customer expectations must be met or exceeded if you want to keep these people to be your customers and start creating loyalty So now let's compare the marketing and sales funnel and then we'll hop right into the specifics of what that might look like For an artist with their music or just brand in general One if you look at this marketing and sales funnel side-by-side You remember from the marketing funnel video you go through awareness consideration conversion loyalty and advocacy so basically this is entire process of let's say a fan discovers you They start to like your music They start to listen to more music after those first few songs they discovered and now they get in with your brand Now they become loyal to you and then they keep this cycle going because they start telling more people about your music All right This is a very much so a content-based thing and we want to keep it super set service level of the marketing funnel Particularly for a musician. They're discovering your music. They're discovering your brand They're consuming your brand and then they're spreading your brand now when we look at the lead generation lead Qualification section of the sales funnel you would actually cut this off as an artist because for the most part Your lead generation and qualification would be taking place in your marketing funnel So that's them discovering your music. That's them Discovering your brand consuming your brand because as you notice there are no sales in the conservative marketing funnel You then combine these as an artist because this is how these work together Sales and marketing go hand-in-hand at the top. There's the marketing funnel and then it leads right into sales Particularly for you as an artist There's different ways people might try to flip this but let's do this traditionally for how it will probably look for an artist You have your awareness fan creation and now there's this loyalty of your fan base and now you start to sell things You don't necessarily have to sell things to only fans Right concert sales and people going to your show can actually be a part of what helps them turn into a fan or even a loyal fan but for the most part what you'll have is These people going through this funnel of becoming fans and now you don't worry about the rest of the sales funnel Now they're fans of your brand when you create products to be available They just sell to your fan base. You're not trying to qualify leads or look for Some leads at that time you are simply just trying to sell to your fan base because they're already qualified as your fan base You don't need to try to go waste all the effort selling to new people making them aware of you and all that stuff So what that looks like when we talk about outbound marketing earlier, you now have inbound marketing Inbound marketing is the fact that as an artist you're creating all this content you put stuff out there You can go to the marketing funnel video to get you know more Attuned to that if you have not watched that video, but once you have that inbound marketing people are self selecting into your process They're self selecting into your funnel because they say hey, I became aware of this artist I like their song or I like their interview. Let me look out for more of them Let me go deeper and deeper into their funnel when you have that outbound stuff Once again, you're putting the effort in you're going out So just imagine if once again you had to call somebody out of the blue or you're trying to go door-to-door or whatever You're getting in people's face. Maybe create some kind of ad or something like that. That's outbound Now the difference is think about Pizza Hut Papa John's whatever piece of place you like to go to have you ever called them for a pizza Have you ever just walked into their doors looking for a pizza? That's inbound They didn't ask you to come there. You got your craving you decided and you came Already ready to buy and that's what you're priming your fan base for when you're building that trust in their brand And they're deciding that they really like you now Lastly, there is a marketing funnel sales funnel combination where you would still use the lead Generation and lead qualification portion of the sales funnel, even though you already have a fan base now Take it back. Once again, you already created fans and we're down to the loyalty standpoint These are your fans that you have But now let's just say you have a product that all of your fan base might not necessarily be able to afford Or all of your fan base might not necessarily be in the right area So some portion of the lead generation could be saying hey, I'm doing a concert That's in LA or I'm just doing my tour So you're trying to make sure fans in a specific area as you go throughout tour are aware of What you have going on so they can then decide to buy your product So now you're targeting specific people within your fan base If you think about Ryan Leslie if you look at the Ryan Leslie video I did he use Superphone his app that a lot of you guys already know about check it out If you don't but he is basically able to filter throughout his fan base And you can see which levels of fans that he has and once a year He has this private party where he's targeting specific fans that spent a certain amount of money on them that has certain behaviors I don't know all of the things that he chooses to target these fans But he chooses them because they'll be more likely to buy and he wants to show some sort of things as well But you can also tell who would be more likely to buy and who's worth targeting in your fan base based on specific Behaviors just like once again if I had a concert on LA my LA fans would be more likely to buy than my New York fans Very simple. Let's keep it at that level, but it applies to a lot of different situations So once again, there's the sales funnel Then there's the combination of the marketing and sales funnel that you'll be using Often once you start to create those fans So a lot of you guys are in the process where y'all are focusing on this marketing funnel And then the combination will be hey then I'm trying to sell to them because I have fans and Then you also have that combination last but not least when you have these fans, but you have specific Experiences they might be exclusive in whatever way they are that you want to target to specific people Within your fan base. There are a lot of different tools to do that and some people hey They just posted on Instagram and hope the right fans find out about it But either way goes take this in people because this is very important in how you view your fan base creation It'll become more and more important as you get more and more fans I know some people are at the very beginning y'all need to solely be focused on that marketing funnel Period now some people are further along y'all have certain levels of fan bases Y'all could really start to take some of these things in and as your fan base grows You might want to reference this video and the marketing funnel video back again and again Just to make sure you're thinking and utilizing and maximizing what you get from your fan base All right now That's my spiel on marketing and sales funnels now Of course, there are a lot of nuances you can get more specific on each and every layer You should do that research on your own if you want to know but other than that ask some questions You know in the comments below we like to answer and discuss questions I would like to know what you guys think and last but not least if you liked this video Go ahead hit that like button if you like it you might as well share it and if you're not subscribed You know what to do here does subscribe button


Welcome to ToffeeTV it is the player ratings Everton Nill, Liverpool Nill a top derby game if you ask me I think it was one of the best derbies for a long while I'm sure Liverpool fans won't agree because you never won it but I think it was one of the most competitive derby games for a while and when I say competitive I don't mean ever just sitting back and hoping Liverpool wouldn't score excuse me it was a game full of chances for both teams and yes Jordan Pickford has been the hero for Everton, Everton had big chances as well and missed a couple real big ones and obviously had a goal disallowed which depending on what way the line is drawn it is offside and I'm not going to sit here grumbling about it although it was a fantastic moment Rob from us yeah we just looked a lot better didn't we we looked a lot better we looked a lot more competitive still got issues in front of goal definitely still got issues in front of goal but it's a clean sheet and we've had to go we've had a real go today I'm going to say real go I don't just mean you know stuck 10 men behind the ball we've asked Liverpool questions and the goal he's made a brilliant save he's made another one off Mopai which we should have scored from and I don't think they liked it open that's what I would say I don't think they liked it open whatsoever let's get into the play of Eaton themselves Jordan Pickford magnificent performance from Jordan today 10 out of 10 performance he gets a 10 from me I mean I don't think I gave him anything else so 10 out of 10 performance the save from Nunes in the first half was incredible the one they tipped onto the bar but then obviously some of the other ones it's the saves where he's pushing it wide he gets that little push motion and pushes it wide and obviously there's one at the end where Salas he's Salas hit the post and he gets his fingers on it now whether the fingers change the trajectory of the of it it doesn't really matter does it because he got his hand on it and but he was phenomenal he was phenomenal today the captain captain and he led by example in his performance and you see how much it meant to him you know Liverpool fans love to have a dig at him and I mean just on Liverpool fans with with was that was there actually any Liverpool fans at the ground today I really I honestly honestly it was like 3,000 mannequins had been shoved in the corner they are one of the worst set of away fans I've ever seen every year they come to Goddison and you do not hear a peep out of them they are some of the worst away fans in existence they are absolutely shocking you don't hear a peep out maybe it's because we're loud and but you just don't hear a peep out of them but Pickford was just just brilliant today and you know that's what you have you that's what you need the top goalkeeper for you need the top goalkeeper to make saves you know their goalkeepers made two billion saves as well so just a fantastic performance from from them I've given my 10 Nathan Patterson probably probably again I keep on saying this every week is best ever in game he was tenacious he was based on a gut to get forward he's found the level of the Premier League now he's developing a real balance to his game he knows he's looking like he knows when to when to put a tackle and when not to pull a tackle him when to intercept just all these little things are starting to lean into his game and he was open down for the whole 90 minutes and he was he had a brilliant game I've given my nine he had a brilliant game and he almost he had one didn't he I think he had a base into the box and had a shot as well I think it took a deflection and went wide for a corner but absolutely brilliant performance again from him just little little tricks as well Robinson came on and he smashed one arm for him to go out for it for a throw and people love all that kind of thing and it's all get right round game he's getting better every game every single game he's got his own song now which is massive and for some people I mean for a lot of people here be our man of the match he's not our man of the match I mean that's just nonsense because the goal he is man of the match and but from the outfielders he probably was our man of the match he was just up and down the just tenacity the tenacity was there it's what a lot of people had seen when he was at at Rangers and play for Scotland those moments thought those moments of wanting to wanting to based up and down the line and have some real energy his crossing led him down a little bit in the first half but once he got into the game once he was up and down he was he was he was absolutely brilliant and on the other side Michael Enko I think Michael Enko had this best game of the season again he was tenacious he wanted everything he wanted to win everything he wanted to be he wanted to be on it he was on it he was making sure he just got little things in the way little a photo ahead or just a block just his body defensively superb today superb I've given him an eight and he was just it was his best game of the season he's looked a little bit off but I think playing left back is suit him a lot more than playing at wing back I don't think wing back suit him naturally but I think and I think playing as a full back it's his game he understands the game a lot better and I thought it was his best game performance of the season Conor Cody I always talk about these probably always talk about these two together Conor Cody and James Tarkowski I've given them both an eight because again leadership and getting the body in the way getting getting the head in the way and I mean Conor Cody where they got more of his goal had been given what a moment for him just I know it's disallow but what a moment for him when you see him score and what it meant to him and if there are any doubters out there about allegiances and all the rest of it well I think you've seen it there there when he scored and I've actually seen people like criticising him for celebrating what what planet are you on what planet are we on when when we can't have that spontaneity to to celebrate the same with the cloud it you know if you're sitting there waiting for it for to see if a goal is given then the game is dead the game is dead if we every decision that is made we wait for to lay fell in a in in a studio somewhere tells you whether it's on side or it's offside football is all about spontaneity and if you celebrate and then the goals taking off you then it's just it's just it's just another thing isn't it we've had goals it happens where you score goals and then the lines once put this flag up it happens it's football it's been happening forever it just is different now because it's VAR and I think that the issue was it takes so long now or that one seems to take so long to get the to get the to get the whether it was on side or offside that that it can't you know it's an it is annoying it is annoying to from that point of view but that's that's just that's just life you roll with that don't you that's football it's about a motion you celebrate and then I get taken off yet whatever I've been taken off as what do we do then that we celebrate again it's nonsense absolute absolute nonsense so I've given them both eight and only one moment I think with the noon I showed that they get caught over the top for a ball I thought the handled everything really well they didn't get bullied and I think that as centre backs the leadership it's just it's just so impressive to see finally see a couple of leaders in the evident team and what it means to them what it means to defend so it's just it's gonna help take the team forward really is Tom Davis I told Tom Davis was brilliant I'll give a monkey again I couldn't care less what anyone else thinks because there's just this bias towards Tom Davis I thought he was excellent I thought he was really unlucky to be taken off I think if I think it just because more of his size because of because of Ghana and and all the rest of it but I thought Tom was great today I thought he was great I thought he broke up the play really well you know we had a few little moments where the you know it he went to do things and doesn't always come off but he's got a head he's got a brain you know he's got a brain and he uses it he doesn't eat today I mean the other day with someone doing something stupid I appreciate that but today use this brain and he just got in there and he broke things up and I thought he had a great game I really did I really did and if people can't see you had a great game then it's just biased it's just biased that you don't like the kid I thought he had a really good game today and then you know taking him off and then bringing bringing bringing Ghana on was brilliant and another day the shot that it's the post goes in I think that's the second time he's hit the post an amazing side Derby and that gone in that gone in for a young lad a blue growing up again it would have just been would have been amazing scenes but it just wasn't meant to be for him but I thought he was brilliant in the mid Liverpool they've got one of the best mid fields in the country by a mile they are that's what makes them so strong you know last season we played two against three in there and the absolutely destroyed us you know I thought for them I thought Harvey Elliot played I don't like the kid particularly but I thought when he had I thought when he had the when he had the ball he's playing some good but really really good balls and it was important to try and shut him down early and I thought Tom Tom's role is to stop them playing that's it that's what his role was and he done it really well today can we do better yes that's what we bought Ghana for but Ghana wasn't available to start so again I don't see the point being there's no there's no need don't be hyper critical about players when they come in and do a job he done a job and he was excellent in the job he done and again if you want to start nitpicking and find the little things oh he was put this and that bollocks he had a really really good game in there and I've seen him playing matey side Darby's where it's been where where the game has passed him by or he's been caught up local like caught up with the with the you know with the emotion and we'll talk about getting caught up with not emotion but rabbit in the headlight in a moment but I thought Tom was great a woby superb as well and again you know got round the pitch lovely little lovely little given goes trying to play clever balls but it's way greater superb I've given him an eight as well it's way greater superb and the the little you know in a three it's gonna really help him move forward and that what he's got going with oh Nana is is is brilliant to be honest absolutely brilliant and it's been you know for him he's starting every game into my field that must be tough for him for where he's been but he's strong and he's getting fitter and he's hung in there with with and that this is the same old Tom and this is the same old own on it you've hung in there okay Liverpool's make little's make up a little midfield was different today it wasn't a strongest I appreciate that but just the way they play and the way they press makes it so hard you know there is you see we've seen this more last year and I think we've seen it moments of the games when they was tiredness or they press at different times in the game they don't always go out it's like the they have like timed intervals of winter press and the force the force the force the ball to go out you know to people to overplay it and stuff like that the harrier and harrier so whatever the make of their three is it's not only the makeup it's how do you play it's how do you train the train to play that way it's really difficult and I thought we I thought we kept that you know our heads briefly today and so and I thought oh well we was a big part of that so I've given my name so Nana and again his best game and everything she had just keeps getting better and better he was he got booked and I think that the booking just sort of meant he had to calm down a little bit in some of the tackle not that he still didn't go in for those boss sliding tackles but he wasn't maybe a times pressing as much as he could because he didn't want to get caught that might but then that changes how he plays and for a player who come in in those two cameos of you know we all know what he played like in those two cameos he settled down amazingly I've given my name to an eight and a half I just thought he was so good so good today and in midfield you know competitive and it's tackling is good it's passing was good and and he just is just getting better and you can see you see what we've been crying out for so long absolutely crying out for his kind his kind of player that kind of player and in the in the midfield so I told you I thought he was I thought he was super been there today and you know as long as we can as long as we can keep that going and then you've got garnets are coming with it and it's it's it's it looks interesting doesn't it looks like looks like we've got different choices we've looked like we've got options possibilities the ability to bring different players on when the Corey comes in you'll have someone who can come on for Oh Nana for his size or or even you know a woby and we've got James Garner was obviously wasn't on the bench today got so many different options on for a team that relied on the Corey and Alan for so long that midfield than those midfield options are huge huge going forward absolutely huge going forward and so I've given on an eight and a half I just thought he's especially for the first made side Barbie he settled down a lot quicker than I honestly thought that he was going to think he settled down massively really quickly you know we spoke about we spoke about about Fellaini when he came in and his first his first abby was his first game and he was all over the place now considering this is Oh Nana's third Premier League game in a week and it shows how far he's come in a week so is it a week is it a week is like his first game with Brentford wasn't it that was his first start and and then you know when Garner comes in he's gonna have that experience he's gonna have that experience now as well so interest and times for everything interest and times and the money great don't all right some good some good runs and some good runs not all not not a massive amount of end product again but the ways times where we just flashed across the box and there's no one in there I did want us to just there's one in the second half and I think we had three in the box and they had the opportunity to go on the outside and he never he went back again and come inside and play to a woman I'm just thinking flashy flashy across see what happens if it hits someone and goes in I want to see more of that but he was all right you and listen where dad again keeps his positional sensory well and I've given a seven and a half but I think I think the next thing that we need to do is upgrade those areas of the pitch we have to upgrade upgrade those those those areas the wide areas that's the next place we need to get more we need to be more clinical in those in those areas so I've given a seven and a half Malpie I've given him a seven and a half decent debut I think he should have scored in the second half I think not to score that the gladus sweet end I actually thought if he'd left it there was someone behind him who had a tapping because I think we had about four then four on four or something like that on that break from a corner so yeah it did all right he worked really hard you wait for a player don't think he's played for a few weeks and he played really hard and it's just so much better when we've got a sense of forward so much better when we've got a sense of forward and he did all right so I've given a seven and a half Anthony Gordon I thought Anthony Gordon was the disappointing part of today I thought I thought he was poor I'll be honest with you I thought he was poor I thought he went and I think he went missing today to be honest I think he was I think he just he was never in the game from the start he never really had the going since Alex Alexander Arnold which again is a disappointment few 50 50s he bottled out of he was one he was one I think we're Malpie put it across the goal and if he just gambled he would score the tapping at the far post in the first half he was nowhere near today he was nowhere near nor at the level at all today I've given a six and he was nowhere nearly which was which was disappointing it was a lovely move that as well from Malpie done a lovely little lovely little move and cross put it right across and all he has to do is gamble all he has to do is gamble which again when we're somewhat scoring goals this in the 17 people have got to gamble that's what that's what natural goal scorers or people who score 10 goals a season do to get at the far post and the the the knocker in for a little simple tapping so I was disappointed him today it's a big opportunity for him to shine and I just don't think he was anywhere near it just it just I don't know he looked like he was breathing out of his ass after 10 minutes he looked just wasn't there wasn't there at all for him today interesting you know just I don't know I don't know I don't know what what it was with him to be honest he has to he has to because we had chances then we had those little half chances like that that if someone taps them in at the far post because he should have been tapping that in you know very much like I mean the goal corner code he scores but obviously it gets disallowed you've got to gamble and there's a sense about gambling and Andy Gordon's not gambling and I don't think the marigold gambles on those chances are not far there and so but it's early days it's early days for certainly for Malpie and the subs gonna I've given I thought he was brilliant when he came on I thought he was absolutely brilliant just just broke the play-up and then give it he was one early on he won it and he smashed it back to Michael Enco and it was like I was like oh because it was like he was smashing it back to a Paris Saint-Germain player who just like take it in his side he smashed it back to him but Michael Enco took it quite well to be honest and because that's what top sides do they just smashed the ball into each other and and you know get on with it but he was brilliant today absolutely but I've given eight actually guys I thought he was he just came on it was like he'd never been away and you can just imagine him sitting in front of that back four with own on and well being front of him that's you know that's what we want to see that's exactly what we want to see and he's got a week now to get you know to get to get more equated with the rest of the team and get that fitness that I don't think he needs I think he fit fit as an absolute fiddle to be honest but he was superb when he came on today and when you need that little moment knowing you want it because the thing was they brought this is they brought the game look at the subs they brought on and that's the difference isn't it you're looking at the bench going oh god this here comes for me now who you know early on changed the game for them he had some big chances had some big chances and he changed and then you've got he changed the fall backs then he brought like jotted on and the and we're bringing we're bringing Dwight MacNeill on and we're bringing and obviously we're bringing Garon they're our only subs and that's the so obviously we worked our bollocks off in that game and late on you're thinking these and that's why the last five minutes were tough the last five minutes were tough because they had fresh legs all over the park did fresh legs a full back did fresh legs up front you know in midfield it's fresh legs everywhere we brought two players on and and that's the difference but to take Tom Davis off and bring Garon I mean that's exactly what you want that's exactly what you want so and hopefully you know next week we might have Dom sitting on the bench and that'll be big for us so yeah Dwight MacNeill came on for for Anthony Gordon and to be fair at all I don't think he ever got into the game he had one shot forever just trying to get on that left foot you had one we were breaking away and he slowed the game down but he was trying to get on the left foot all the time he had the one shot that if you watch it again strikes the hand of Milne or the arm of Milne and I tell you what in another country that's a penalty maybe not in this country but in another country that's a handball an absolute handball so but he never quite gone into the play I don't want to slag him off he just didn't get into the pace of the game whatsoever and he looked knackered he just couldn't get up to speed with the pace of the game he was he was just he was just never he was just he looked tired after five minutes ten minutes or whatever he was on for because he just the pace of the game was ferocious absolutely ferocious but we take a point take a point that's our fourth pointer is that four four draws in a row it is it was yeah four draws in a row five one beating we go to Arsenal and then we've got West Ham tough games if we can start conveying those chances we can get wins we can get wins so we'll have to see what happens but yeah but I mean listen I'm not saying you're going on made up with the point and made up with the performance and made up to see an evidence team not fall to bits under pressure against Liverpool that's what I'm happy about that's the positives I'm disappointed we never won and that's three games in a row or all four games I'm disappointed we never won and I'm disappointed we never got something against Villa and I'm disappointed we never got something Chelsea so I'm I'm disappointed I'm disappointed every game that we haven't got something out of it more out of it sorry so we you know we sat in in December and we sat in December and we we'd been beaten for one and there was today was a completely different team I think there's only one one play one outfield player who started so it's keep working hard keep showing up put them performances against other teams and we'll get was up we just need need to get that face win under our belt and then go from there but I think I think anyone and this is like you know not fans as such but this is the media the media talking about Frank Lampard well I think there you go today there you go shows everybody that the fans are behind them and he's getting he's changing things to the way he wants to do things and if we could just add goals to this team which I know is the hardest thing and I know that's why people were disappointed on on on Thursday that we never got another one but if we can start adding goals then we'll we'll we'll shoot up that table quickly so there you go good point on to the next one now make sure to check out if you haven't already my glad to see the action make sure to check out Bazaar's videos and we'll be doing the final word on Monday live over on toffee TV premiere also the press conference check out the aftermatch press conference that'll be available on toffee TV don't you won't find that anywhere else really well you might have to have a good luck but you know you'll be on be on the channel in the next couple of hours so there you go thank you for watching and I'll see you later