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5. Protein Techniques

Peptide Mass Fingerprinting


Peptide Mass Fingerprinting

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in this video, we're going to talk about peptide mass fingerprinting, so peptide mass fingerprinting could be abbreviated with PMF. And really, it's just a technique that we use toe identify unknown proteins. And the way that it works is that it uses the mass spectrometry spectrums from very large computer databases in order to identify the unknown proteins. And so, if we have an unknown protein sample, we know that we could take a little bit of this unknown protein sample and subjected to tandem mass spectrometry in order to acquire an actual spectrum of the unknown protein. And then we can compare this actual spectrum of the unknown protein to the spectrums of all known proteins that are stored in computer databases until, ah, match is found between the actual spectrum of the unknown protein and all of the spectrum's from all of the known proteins on DSO, once a match has been found, were able to identify the unknown protein. Now, usually it's on lee necessary toe. Analyze very small portions of the unknown protein in order to identify it. So normally it's not necessary. Toe. Take the time to analyze the entire unknown protein to identify and that speeds up the process and makes it quicker. Now, an actual limitation to peptide mass fingerprinting is that the database must already include the protein in order to identify it. And so, if the database does not already include the unknown protein, then we've discovered a brand new protein that has never been discovered before. And then we can take the time toe fully sequence that unknown protein, and then we can enter that unknown protein sequence into the database so that later down the line, if anyone ever extracts that same unknown protein, they can use peptide mass fingerprinting to quickly identify. So let's take a look at our example down below peptide mass fingerprinting to clear up this whole idea better. All right, So which will notice is we've got these two brackets here. We've got this first bracket right here and this second bracket over here. Now, the first bracket and yellow is essentially the entire tandem mass spectrometry, uh, process that we already covered in our previous lesson videos. And the second bracket on the far right is referring to the peptide mass fingerprinting, which is the main focus of this video and recall that peptide mass fingerprinting can be abbreviated with just Pete M. F. And so, with Tana Mass spectrometry, we know that we can start with an already purified protein, and here we have an unknown protein that we're starting with and weaken, subjected to fragmentation with a chemical or a protease, and will generate a bunch of different protein fragments. And then we're going to ionized these protein fragments and subject them to our first mass spectrometry. And we know that this first mass spectrometry is really acting like a filter to select for very particular peptide fragments to continue forward in the process. And so, essentially here it's showing an actual spectrum for the first mass spectrometry, and that's totally fine. Unacceptable. It's possible that that could happen here, but we know that with 10 a mass spectrometry Onley very particular peptide fragments are gonna be selected to move forward in the process. And so, the first time here that we perform Tana Mass spectrometry, we can move this particular fragment forward in the process. We know that we're going to subject it to ah, collision cell where it's going to fragment even further and then subjected to a second mass spectrometry the M S M s to generate a spectrum of this particular peptide fragment. And then we could do the same with a different fragment on a different round and generate this spectrum here. And then, of course, in a different round of Tana mass spectrometry weaken, subject this fragment to move forward and generate that spectrum. So here we've got three different spectrums that we've subjected to tandem mass spectrometry or M s m s. And really, these spectrums here that are generated from M S, M s are so unique and specific that they really act like fingerprints. And so up above here, I've provided a fingerprints so that you guys can see that the M SMS spectrums that air generated really act like fingerprints. And just like we can use your fingerprint toe, identify you in a large database of fingerprints. We can use these spectrums here and enter them into a computer database and then have the computer search, uh, the entire computer database until it finds a match for these spectrums. Now the spectrum's that are stored in the computer database are also linked to protein sequences. And so once these spectrums that have been entered into the computer database are matched to a sequence that's found in the computer database. Then we've obtained the actual protein sequences for these particular peptide fragments that we selected to move forward. And once we've identified, once we've matched the spectrum to a sequence, then it's pretty easy. Toe identified the actual sub units, and once we've identified the actual sub units that these fragments came from, it's pretty easy to identify the unknown protein that we originally started off with. And so, essentially, that is the process of peptide mass fingerprinting, uh, using tandem mass spectrometry to generate very unique mass spectrums and then entering these unique mass spectrums into a computer database until we find a match. And then once we find a match, we identify the sub units and identify the protein. So this year concludes our lesson on peptide mass fingerprinting, and our next video will be able to talk about other ways to sequence a protein. And so this concludes this video and I'll see you guys in our practice videos

Peptide mass fingerprinting (PMF) is a method used to determine the sequence of an unknown peptide. In the sequencing of peptides by PMF, tandem mass spectrometry fragments peptides into smaller molecules to obtain the mass spectrum, which acts like a fingerprint & can be compared to theoretical spectrums in a database to derive the amino acid sequence. Mainly which types of bonds are broken to fragment the peptide & generate tandem mass spectra data?


Explain how a 5-residue amino acid sequence could uniquely identify a 200-residue protein via PMF. 

Hint #1: Compare the number of pentapeptides in the 200-residue protein to the total number of all possible pentapeptide sequences.

Hint #2: 

Hint #3: 

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