Detection of modified peptides in enzymatic digests by capillary liquid chromatography/electrospray mass spectrometry...

A method for the identification of multiple covalent protein modifications in enzymatic protein digests by specific marker ion signals in a single analysis is described. This method is based on the combined strengths of capillary liquid chromatography (microLC) to purify, concentrate, and resolve complex mixtures and electrospray mass spectrometry (ESI-MS) to selectively and sensitively detect ions. A variety of modification-specific marker ions can be generated using a programmable skimmer collision-induced dissociation (sCID) acquisition routine, which allows for flexibility in the (i) number of marker ions monitored under single-ion monitoring conditions, (ii) selection of optimal polarity for both marker ions and molecular ions, (iii) use of variable dwell times for marker ions, and (iv) selection of optimal sCID offset. Using this combined method of microLC/ESI/sCID-MS, phosphorylated, sulfated, acrylamide-modified, and glycosylated peptides were identified in a model enzymatic digest at 200 fmol. The capability of reversed-phase LC to resolve isomeric compounds which cannot be identified by low-energy CID underscores the utility of this combined method. Further capabilities of this technique are demonstrated by the analysis of biologically important proteins.