Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de novo sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry.
A simple mass spectrometric method to sequence a recombinant phosphoenolpyruvate carboxykinase of known structure and a novel variant of unknown structure isolated from Anaerobiospirillum succiniciproducens and Actinobacillus succinogenes 130Z, respectively, was evaluated. The proteolytic digests of the proteins were each chemically derivatized at the N-terminus by addition of a tris(trimethoxyphenyl)phosphoniumacetyl (TMPP(+)-Ac) group to produce peptides with a fixed positive charge. The derivatized digests were then partially separated by reversed-phase high-performance liquid chromatography. The fractions collected were subjected to matrix-assisted laser desorption/ionization post-source decay (MALDI/PSD) mass spectrometric analysis. The resulting spectra are sufficiently simple to allow the sequence to be read directly without extensive interpretation. This is in contrast to spectra of underivatized peptides obtained by MALDI/PSD or conventional tandem mass spectrometry, where full sequence interpretation can be challenging. Aided with a set of very simple established rules, it was shown that the sequence of TMPP(+)-Ac derivatives can be derived strictly from predictable fragment ion series. In most cases, this is sufficient to determine extensive, unambiguous, peptide sequences de novo. The partial sequence (35%) of the unknown phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes 130Z was obtained entirely by the mass spectrometric method evaluated here, which provided the basis for evaluating homology and for the design of oligonucleotide probes for cloning the corresponding gene.
Shen
T L
TL
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Huang
Z H
ZH
Laivenieks
M
M
Zeikus
J G
JG
Gage
D A
DA
Allison
J
J
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D055672:Static Electricity
D010446:Peptide Fragments; D011994:Recombinant Proteins; D010729:Phosphoenolpyruvate Carboxykinase (GTP)