Analytical Sciences

Exopeptidase Degradation for the Analysis of Phosphorylation Site in a Mono-phosphorylated Peptide with Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Nariyasu MANO,*  Setsuko IIJIMA,* Kie KASUGA,* and Junichi GOTO*^,**

*Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba, Sendai 980-8578, Japan
**Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba, Sendai 980-8574, Japan

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 β-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.

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