학술논문
C-Terminal sequencing of protein by MALDI mass spectrometry through the specific derivatization of the α-carboxyl group with 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide.
Document Type
Article
Source
Subject
*C-terminal binding proteins
*MATRIX-assisted laser desorption-ionization
*CARBOXYL group
*PHOSPHONIUM compounds
*BROMIDES
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Language
ISSN
1618-2642
Abstract
We present here an approach to C-terminal sequencing of proteins by the procedure consisting of the following: (1) derivatization of the C-terminal α-carboxyl group with 3-aminopropyltris(2,4,6-trimethoxyphenyl)-phosphonium bromide (TMPP-propylamine) through oxazolone chemistry, (2) enzymatic proteolysis of the TMPP-derivatized protein, and (3) MALDI-MS/MS analysis of the peptide mixture, in which the C-terminal peptide incorporating the TMPP group is preferably detected. In this protocol, it is possible to choose any endoproteinase such as trypsin, GluC, and AspN for digestion so that a C-terminal peptide with length appropriate for mass spectrometric sequencing could be generated. The peptide labeled with TMPP-propylamine at the C terminus tends to exhibit y-type ions in MS/MS spectra, allowing manual sequence interpretation with the simplified fragmentation pattern. The efficacy of the method was verified with five proteins, which demonstrated that the C-terminal peptides were readily distinguishable by their peak intensity and characteristic mass signature peak in MALDI-PSD analysis. [ABSTRACT FROM AUTHOR]