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.
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.
Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
Chihiro
C
Nakajima
Hiroki
H
Kuyama
Takashi
T
Nakazawa
Osamu
O
Nishimura
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D010856:Columbidae; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013552:Swine
D011506:Proteins