Protein identification and tracking in two-dimensional electrophoretic gels by minimal protein identifiers.
Protein identification by matrix-assisted laser desorption/ionization mass-spectrometry peptide mass fingerprinting (MALDI-MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low-molecular-mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI-MS-based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two-dimensional gel electrophoresis (2-DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI-MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein-specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF-Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2-DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.
Mattow
Jens
J
Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany. mattow@mpiib-berlin.mpg.de
Schmidt
Frank
F
Höhenwarter
Wolfgang
W
Siejak
Frank
F
Schaible
Ulrich E
UE
Kaufmann
Stefan H E
SH
D001426:Bacterial Proteins / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry; D018832:Molecular Chaperones; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D010455:Peptides; D020543:Proteome; D014357:Trypsin; D010444:Peptide Elongation Factor Tu