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1、MS质谱测序原理Peptide sequencing by mass spectrometrylab-5102 reporter:feng liIntroduction: 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 _amp; ine_pensive form of M
2、S(MALDI) can be done by a moderately skilled operatorCons need sequence in the database generally found to only be avout 40% effectiveMS/MS vs PMF Advantages provides precise sequencespecific data more informative than PMF methods(_gt;90%) can be used for de-novo sequencing can be used to ID posttra
3、nslatinal modificationsDisadvantages requires more handling, refinement and sample manipulation requires more e_pensive and complicated equipment(high resolution) requires high level e_pertise,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 M
4、S) b-e_tend from N-terminal y-e_tend from C-terminal a-used as a diagnostic for b ions separated by C=O 28ub ions:sample peptide GLSDGEWQQVLNVWGKThe figure shows the first si_ 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 ionsb
5、5 Attention:b ion intensity may drop when the ne_t residue is P, G or also H, K, and R,Handling rules for sequencingLoss of Ammonia and Wat erl.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.
6、Spectral Intensity Rules 1.b ion intensity will drop when the ne_t 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 encounte
7、ring aspartic acid in a sequence, the ion series can die outAmino Acid Composition It is possible to observe immonium ions at the low end of the spectrum that can give a clue to the amino acciodmposition of a peptide.Isobaric Mass1.leu_amp;Ile,then label it _ 2.Lysine and Glutamine have near isobari
8、c masses, 128.09496 and 128.05858 respectively 3. two residues will nearly equal the mass of a single residue. e.g-Asn=Gly-GlyMore 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 s
9、pectrum, observe 147 for K or 175 for R. 4.once you know the mass of a b ion: y = (M+H)1+ - b +1The pro to col following the b ion seriesa t rip tic petide f low end147 indicates a lys;175 indicates an argformula: (M+H)l+ - yl+1 = pen ultimate b ionItake the smallest AA jump , look for the ne_t b io
10、nfind the corresponding y ionTryptic peptides may have a more prominent y ion series.The first step:(M+H)1+ - AA = penultimate y ionor (M+H)1+ - observed ion = AA ; the following steps are same as b ion series!DeNovoE_ercise Herearethehints:1.Thisisatrypticpeptideending in K or R. 2.The Parent M+H1+ mass is 1379.4 3.Since this is ion trapdata, mass accuracy should be within 0.8uFirst,find the penultimate b ion.Then, high mass to low mass by the smallest jump!
