Mapping and sequence-specific identification of phosphopeptides in unfractionated protein digest mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
We have demonstrated a procedure for the rapid (minutes), sensitive (less than pmol), and sequence-specific identification of phosphopeptides in unfractionated digests of phosphoproteins using matrix-assisted UV laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. The mass-dependent identification of one specific 13-residue phosphopeptide (S105-K117), observed among the 153 possible trypsin digest fragments of human beta-casein (211 residues), was confirmed by amino acid sequence analysis of the 33P-labeled peptide after isolation by reverse-phase HPLC. MALDI-TOF was also used to monitor the rate and extent to which an 18-residue N-terminal beta-casein peptide (R1-K18) was phosphorylated in vitro. These results demonstrate that MALDI-TOF may be used (i) to facilitate the identification of sequence-specific sites of protein phosphorylation and dephosphorylation, (ii) to monitor protein and peptide phosphorylation and dephosphorylation reaction rates, even in complex unfractionated mixtures, (iii) to determine the minimum primary structure necessary for the phosphorylation of specific protein surface domains, and (iv) to evaluate the effects of intact protein phosphorylation and dephosphorylation on susceptibility to subsequent proteolytic events.
Yip
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TT
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
Hutchens
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TW
D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007477:Ions; D007834:Lasers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation
D002364:Caseins; D007477:Ions; D010748:Phosphopeptides