An approach to locate phosphorylation sites in a phosphoprotein: mass mapping by combining specific enzymatic degradation with matrix-assisted laser desorption/ionization mass spectrometry.
A rapid, picomole-scale method is described to locate phosphorylation sites in phosphoproteins by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with enzymatic modification of the analyte. There are three steps to locate phosphorylation sites in a phosphoprotein: (i) degradation of the phosphoprotein into small peptides by specific enzymatic or chemical reactions; (ii) identification of the phosphopeptides by -80 (or multiples of -80)-Da mass shifts in the mass spectra after dephosphorylation with alkaline phosphatase; (iii) location of the phosphorylation sites by mass mapping. As the size of the protein increases, it is advantageous to fractionate the mixture by HPLC and analyze each fraction by MALDI-TOF-MS. To perform mass mapping, the primary structure of the protein must be known. Bovine beta-casein was analyzed by this method. The conclusions about the specific phosphorylation sites of bovine beta-casein from our data coincide with previously reported results. From calculations, it is found that a mass spectrometer with 0.1% mass accuracy is sufficient, for mass mapping, to identify completely or partially digested tryptic peptides in the mass range of 100-8000 Da from bovine beta-casein (MW 23,983).
Liao
P C
PC
Department of Chemistry, Michigan State University, East Lansing 48824.
Leykam
J
J
Andrews
P C
PC
Gage
D A
DA
Allison
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D012680:Sensitivity and Specificity
D002364:Caseins; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins