Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry.
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 (MS(n), n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI-MS(n) is demonstrated by the analysis of tryptic digests of alpha- and beta-casein and in-gel tryptic digests of beta-casein.
Cao
P
P
Protein Chemistry Department, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, USA.
Stults
J T
JT
D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry; D002846:Chromatography, Affinity; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009862:Online Systems; D010449:Peptide Mapping; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry*; D014357:Trypsin
D002364:Caseins; D011506:Proteins; D014357:Trypsin