MS质谱测序原理.ppt

Peptide sequencing by mass spectrometry lab-5102 reporter:feng li Introduction: there is two main approaches for protein sequencing a) 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 sequence-specific data more informative than PMF methods(90%) can be used for de-novo se-quencing can be used to ID posttrans-latinal modifications De Novo Peptide Sequencing Tutorial- how to find the b,y peak? What are b,y and a ions(the most common in MS) b ions:sample peptide GLSDGEWQQVLNVWGK b and y ions Handling rules for sequencing Spectral Intensity Rules 1.b ion intensity will drop when the next residue is P, 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 Mass 1.leuIle,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 series Tryptic peptides may have a more prominent y ion series. The first step:(M+H)1+ - AA = penultimate