2019年最新-Identifying-Proteins-with-Tandem-Mass-Spectrometry---Lectures:识别和串联质谱蛋白质-讲座-
Interpreting MS/MS Proteomics Results,Brian C. Searle Proteome Software Inc. Portland, Oregon USA Brian.SearleProteomeSoftware NPC Progress Meeting (February 2nd, 2019),The first thing I should say is that none of the material presented is original research done at Proteome Software,but we do strive to make the tools presented here available in our software product Scaffold. With that caveat aside,Illustrated by Toni Boudreault,Organization,This is an foremost an introduction so were first going to talk about,Then were going to talk about the motivations behind the development of the first really useful bioinformatics technique in our field, SEQUEST.,This technique has been extended by two other tools called X! Tandem and Mascot.,Were also going to talk about how these programs differ,and how we can use that to our advantage by considering them simultaneously using probabilities.,Identify,SEQUEST,X! Tandem/Mascot,Differ Combine,how you go about identifying proteins with tandem mass spectrometry in the first place,So, this is proteomics, so were going to use tandem mass spectrometry to identify proteins- hopefully many of them, and hopefully very quickly.,A,A,I,K,G,K,I,D,V,C,I,V,L,L,Q,H,K,A,E,P,T,I,R,N,T,D,G,R,T,A,Start with a protein,And to use this technique you generally have to lyse the protein into peptides about 8 to 20 amino acids in length and,A,A,I,K,G,K,I,D,V,C,I,V,L,L,Q,H,K,A,E,P,T,I,R,N,T,D,G,R,T,A,Cut with an enzyme,A,A,I,K,G,K,I,D,V,C,I,V,L,L,Q,H,K,A,E,P,T,I,R,N,T,D,G,R,T,A,Select a peptide,Look at each peptide individually.,We select the peptide by mass using the first half of the tandem mass spectrometer,A,E,P,T,I,R,H2O,Impart energy in collision cell,The mass spectrometer imparts energy into the peptide causing it to fragment at the peptide bonds between amino acids.,M/z,Intensity,A,E,P,A,A,E,A,E,P,T,72.0,201.1,298.1,399.2,Measure mass of daughter ions,The masses of these fragment ions is recorded using the second mass spectrometer.,M/z,Intensity,A,E,P,T,I,R,B-type Ions,H2O,72.0,129.0,97.0,101.0,113.1,174.1,These ions are commonly called B ions, based on nomenclature you dont really want to know about,But the mass difference between the peaks corresponds directly to the amino acid sequence.,M/z,Intensity,A,E,P,T,I,R,B-type Ions,H2O,72.0,129.0,97.0,101.0,113.1,174.1,A-0,AE-A,AEP -AE,AEPT -AEP,AEPTI -AEPT,AEPTIR -AEPTI,For example, the A-E peak minus the A peak should produce the mass of E.,You can build these mass differences up and derive a sequence for the original peptide,This is pretty neat and it makes tandem mass spectrometry one of the best tools out there for sequencing novel peptides.,So, it seems pretty easy, doesnt it?,But there are a couple confounding factors.,For example,M/z,Intensity,A,E,P,T,I,R,B-type Ions,H2O,CO,CO,CO,CO,CO,CO,B ions have a tendency to degrade and lose carbon monoxide producing,M/z,A,E,P,T,I,R,A-type Ions,H2O,CO,CO,CO,CO,CO,CO,A ions.,Furthermore,M/z,Intensity,R,I,T,P,E,A,Y-type Ions,H2O, The second half are represented as Y ions that sequence backwards.,And, unfortunately, this is the real world, so,M/z,Intensity,R,I,T,P,E,A,Y-type Ions,H2O, All the peaks have different measured heights and many peaks can often be missing.,M/z,Intensity,R,I,T,P,E,A,H2O,B-type, A-type, Y-type Ions,All these peaks are seen together simultaneously,and we dont even know,M/z,Intensity,What type of ion they are, making the mass differences approach even more difficult.,Finally, as with all analytical techniques,M/z,Intensity,Theres noise,producing a final spectrum that looks like,M/z,Intensity,.This, on a good day.,And so its actually fairly difficult to,M/z,Intensity,72.0,129.0,97.0,101.0,113.1,174.1,A,E,P,T,I,R,H2O, compute the mass differences to sequence the peptide, certainly in a computer automated way.,So the community needed a new technique.,Now, it wasnt all without hope,Known Ion Types,B-type ions A-type ions Y-type ions,We knew a couple of things about peptide fragmentation.,Not only do we know to expect B, A, and Y ions, but,Known Ion Types,B-type ions A-type ions Y-type ions B- or Y-type +2H ions B- or Y-type -NH3 ions B- or Y-type -H2O ions, We also know a couple of other variations on those ions that come up.,We even know something about the,Known Ion Types,B-type ions A-type ions Y-type ions B- or Y-type +2H ions B- or Y-type -NH3 ions B- or Y-type -H2O ions,100% 20% 100% 50% 20% 20%, likelihood of seeing each type of ion,where generally B and Y ions are most prominent.,If we know the amino acid sequence of a peptide, we can guess what the spectra should look like!,So its actually pretty easy to guess what a spectrum should look lik