Peptide fragmentation
View Patent ↗The present invention relates to a method of identifying a polypeptide using a class of novel, water-stable reagents. More specifically, the method according to the invention comprises the steps of (a) derivatization in an aqueous solution of the N-terminus of the polypeptide, or the N-termini of one or more peptides of the polypeptide, with at least one acidic reagent which comprises a sulfonyl moiety coupled to an activated acid moiety to provide one or more peptide derivatives; (b) analyzing at least one such derivative using a mass spectrometric technique to provide a fragmentation pattern; and (c) interpreting the fragmentation pattern. Furthermore, the present invention also relates to a kit for identifying a polypeptide by a mass spectrometric technique, which kit comprises at least one acidic reagent comprising a sulfonyl moiety coupled to an activated acid moiety in a container together with suitable means for analysis of the fragmentation pattern obtained.
1. A method of identifying a polypeptide, which method comprises the steps of
(a) derivatizating, in an aqueous solution, the N-terminus of the polypeptide, or the N-termini of one or more peptides of the polypeptide, with at least one acidic reagent containing a sulfo moiety coupled to an ester moiety to provide one or more peptide derivatives, which reagent exhibits a half-life in aqueous solution of not less than 10 minutes at room temperature, to prepare one or more derivatives;
(b) analyzing at least one said derivative using a mass spectrometric technique to provide a fragmentation pattern; and
(c) interpreting the fragmentation pattern obtained to identify the polypeptide;
wherein said at least one acidic reagent is an N-hydroxysuccinimide ester of the compound of the formula
wherein Y is a spacer which contains aliphatic and/or aromatic fragments and may optionally include additional sulfonic acids.
2. The method according to claim 1 , wherein the acidic reagent has a pKa of less than about 2 when coupled to the polypeptide.
3. The method according to claim 1 , wherein the mass spectrometric technique used in step (b) is matrix-assisted laser desorption ionization (MALDI) mass spectrometry.
4. The method according to claim 1 , wherein the mass spectrometric technique used in step (b) is electrospray ionization (ESI).
5. The method according to claim 1 , wherein in step (c), the fragmentation pattern is interpreted using a software program or database.
6. The method according to claim 1 , wherein all the steps are conducted as part of an automated or semi-automated procedure.
7. The method according to claim 1 , wherein the acidic reagent comprises a 3-sulfopropionic acid N-hydroxysuccinimide ester.
8. The method according to claim 1 , wherein the acidic reagent comprises a 2-sulfobenzoic acid N-hydroxysuccinimide ester.
9. The method according to claim 1 , wherein the polypeptide has been obtained by enzymatic digestion.
10. The method according to claim 9 , wherein the enzyme is trypsin.
11. The method according to claim 1 , which further comprises a step of protecting lysine residues prior to the derivatizating step.