Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry

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Abstract
On‐line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization‐mass spectrometry (IMAC/CE/ESI‐MS) offers selective preconcentration of phosphorylated peptides with identification of the phosphorylated amino acid(s). The preconcentration provides low concentration limits of detection and capillary electrophoresis separates the peptides. Recently, we reported a fast, simple, and sensitive on‐line IMAC/CE/ESI‐MS/MS method for the determination of phosphopeptides at low‐pmole levels. That work is expanded here by use of multiple stage tandem mass spectrometry (MSn, n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI‐MSn is demonstrated by the analysis of tryptic digests of α‐ and β‐casein and in‐gel tryptic digests of β‐casein. Copyright © 2000 John Wiley & Sons, Ltd.