Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 404, Issue 1, Pages 125-132Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-012-6093-5
Keywords
Bioanalytical methods; Mass spectrometry/ICP-MS; Spectroscopy/instrumentation
Funding
- Ministry of Education, Culture, Sports, Science and Technology of Japan [22510230]
- Grants-in-Aid for Scientific Research [24510296, 22510230, 22650218] Funding Source: KAKEN
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We present here an approach to C-terminal sequencing of proteins by the procedure consisting of the following: (1) derivatization of the C-terminal alpha-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.
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