MS质谱测序原理

Peptide sequencing by mass spectrometry

lab-5102 reporter:feng li

Introduction: there is two main approaches for protein sequencinga) Peptide mass finger printing(PMF),based on a huge peptide databse b) Mandem mass spectrometry(MS/MS)

Pros and cons of PMF Pros robust & inexpensive form of MS(MALDI) can be done by a moderately skilled operator

Cons need sequence in the database generally found to only be avout 40% effective

MS/MS vs PMF Advantages provides precise sequencespecific data more informative than PMF methods(>90%) can be used for de-novo sequencing can be used to ID posttranslatinal modifications

Disadvantages requires more handling, refinement and sample manipulation requires more expensive and complicated equipment(high resolution) requires high level expertise,both handling and software(e.g.Mascot)

how to find the b,y peak?

What are b,y and a ions(the most common in MS)

● b--extend from N-terminal ● y--extend from C-terminal ● a--used as a diagnostic for b ions separated by C=O 28u

b ions:sample peptide GLSDGEWQQVLNVWGK

The figure shows the first six b ions in a little bit more detail. The b ion m/z value is basically the mass of the peptide minus OH, or -17u.

b and y ions

b5 Attention:b ion intensity may drop when the next residue is P, G or also H, K, and R,

Handling rules for sequencing●Loss of Ammonia and Water1.y and b ion containing residues R, K, Q, and N may appear to lose ammonia, -17. 2.y and b ion containing residues S, T, and E may appear to lose water, -18.

Spectral Intensity Rules 1.b ion intensity will drop when the next residue isP, G or also H, K, and R 2.Internal cleavages can occur at P and H residues. 3.When a cleavage appears before or after R, the 17 (loss of ammonia) peak can be more prominent than the corresponding y or b ion 4.When encountering aspartic acid in a sequence, the ion series can die out

Amino Acid Composition It is possible to observe immonium ions at the low end of the spectrum that can give a clue to the amino acid composition of a peptide.

Isobaric Mass1.leu&Ile,then label it X 2.Lysine and Glutamine have near isobaric masses, 128.09496 and 128.05858 respectively 3. two residues will nearly equal the mass of a single residue. e.g---Asn=Gly-Gly

More rules 1.start at the high mass end of the spectrum 2.the region 60 u below the parent mass can be puzzled by multiple water and ammonia losses. 3.at the low end of the spectrum, observe 147 for K or 175 for R. 4.once you know the mass of a b ion: y = (M+H)1+ - b +1

The protocol following the b ion seriesa triptic petide → low end→147 indicates a lys;175 indicates an arg

↓formula: (M+H)1+ - y1+1 = penultimate b ion

↓take the smallest AA jump , look for the next b ion

↓find the corresponding y ion

Tryptic peptides may have a more prominent y ion series.The first step:(M+H)1+ - AA = penultimate y ion

or (M+H)1+ - observed ion = AA ; the following steps are same as b ion series!

De Novo Exercise Here are the hints: 1.This is a tryptic peptide ending in K or R. 2.The Parent [M+H]1+ mass is 1379.4 3.Since this is ion trap data, mass accuracy should be within 0.8u

First,find the penultimate b ion.

Then, high mass to low mass by the smallest jump!

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