Peptide Mass Fingerprinting - Video Tutorials & Practice Problems
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Peptide Mass Fingerprinting
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Video transcript
In this video, we're gonna talk about peptide mass fingerprinting. So peptide mass fingerprinting can be abbreviated with PMF. And really it's just a technique that we use to 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 can take a little bit of this unknown protein sample and subject it 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 a match is found between the actual spectrum of the unknown protein and all of the spectrums from all of the known proteins. And so once a match has been found, we're able to identify the unknown protein. Now, usually, it's only necessary to analyze very small portions of the unknown protein in order to identify it. So normally, it's not necessary to 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 to peptide mass fingerprinting is that the database must already include the protein in order to identify it. 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 to 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 mass fingerprinting to quickly identify it. So let's take a look at our example down below of peptide mass fingerprinting to clear up this whole idea better. Alright. So what you'll notice is we've got these two brackets here. We've got this first first bracket right here and this second bracket over here. Now, the first bracket in yellow is essentially the entire tandem mass spectrometry, 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 we we know that we can start with an already purified protein. And here, we have an unknown protein that we're starting with. And we can subject it to fragmentation with a chemical or a protease, and, we'll generate a bunch of different protein fragments. And then we're going to ionize 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 and acceptable. It's possible, spectrometry. And that's totally fine and acceptable. It's possible, that that could happen here. But we know that, with tandem mass spectrometry, only very particular peptide fragments are going to be selected to move forward in the process. And so the first time here that we perform tandem mass spectrometry, we can move this particular fragment forward in the process. We know that we're going to subject it to, a collision, cell, where it's going to fragment even further, and then subject it to a second mass spectrometry, the MSMS, to generate a spectrum of this particular peptide fragment. And then we can 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 tandem mass spectrometry, we can subject this fragment to move forward and generate that spectrum. So here we've got 3 different spectrums that we've subjected to tandem mass spectrometry, or MSMS. And really these spectrums here that are generated from MSMS are so unique and specific that they really act like fingerprints. And so up above here, I've, provided the fingerprints, so that you guys can see that the MSMS spectrums that are generated really act like fingerprints. And just like we can use your fingerprint to identify you and a large database of fingerprints, we can use these spectrums here and enter them into a computer database, and then have the computer search, the entire computer database until it finds a match for these spectrums. Now, the spectrums 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 to identify the actual subunits. And once we've identified the actual subunits 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. 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 a match, we identify the subunit and identify the protein. So this here concludes our lesson on peptide mass fingerprinting, and in our next video, we'll 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 Yes.
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Problem
Problem
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?
A
Carbon-carbon bonds within the amino acid R-groups.
B
Disulfide bonds.
C
Omega bonds.
D
Phi bonds.
E
Psi bonds.
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Problem
Problem
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.