Exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide with matrix-assisted laser desorption/ionization mass spectrometry.
The utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with a peptide ladder sequencing method employing exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide is investigated. MALDI-TOFMS analysis of time-dependent exopeptidase digestion using carboxypeptidase W and aminopeptidase M of the mono-phosphorylated 33-48 fragment isolated from a beta-casein tryptic digestion mixture allowed for the sequencing analysis from both the C-terminus and N-terminus. Negative ion detection MALDI-TOFMS made it possible to clearly measure the peptide ladder of mono-phosphorylated peptide by the strong negative charge localized at the phosphoric acid group. Since exopeptidase activity was suppressed by the existence of a phosphorylated amino acid residue, the termination exopeptidase degradation therefore suggested the existence of a phosphorylated amino acid residue at that site. This peptide ladder sequencing method using exopeptidases was effective for the identification of the site of a phosphorylated amino acid residue by a simple MALDI-TOFMS analysis in the negative ion detection mode.
Mano
Nariyasu
N
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba, Sendai 980-8578, Japan. n-mano@mail.pharm.tohoku.ac.jp
Iijima
Setsuko
S
Kasuga
Kie
K
Goto
Junichi
J
D000469:Alkaline Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D000626:Aminopeptidases; D001665:Binding Sites; D002268:Carboxypeptidases; D002364:Caseins / Q000737:chemistry; D020689:Exopeptidases / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D002364:Caseins; D010446:Peptide Fragments; D010748:Phosphopeptides; D000469:Alkaline Phosphatase; D002268:Carboxypeptidases; D020689:Exopeptidases; D000626:Aminopeptidases; D014357:Trypsin