Protein identification and tracking in two‐dimensional electrophoretic gels by minimal protein identifiers

Abstract

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.

Biological Variation of the Platelet Proteome in the Elderly Population and Its Implication for Biomarker Research

“…Internal calibration was performed using the singly charged peptides from tryptic autodigestion at m/z 659.38, 805.42, 1153.57, and 2163.06 (monoisotopic masses). The lists of monoisotopic m/z values (signal to noise ratio of a signal had to be above 3:1, and a plausible isotopic pattern had to be observable) for peptide mass fingerprint (PMF) searches were manually derived from unsmoothed mass spectra without base-line subtraction, omitting those values related to tryptic autodigestion, contamination from keratins (14), and matrix cluster ions (15). Peak annotation was carried out automatically using software provided by the instrument manufacturer (Launchpad, version 2.7.1.20060929; Shimadzu Biotech).…”

Decimal Place Slope, A Fast and Precise Method for Quantifying 13C Incorporation Levels for Detecting the Metabolic Activity of Microbial Species

“…decimal places as described elsewhere (24). The uniform slope has its origin in similar distributions of carbon (C) and nitrogen (N) in each of the peptides, independent of their lengths (28).…”

“…Calculation of 13 C Incorporation Levels of Experimentally Derived Peptides-We used a peptide database of Mycobacterium tuberculosis (initially containing Ͼ9 ϫ 10 4 tryptic peptide sequences with lengths of 2-40 amino acids) as a reference (24). In the first step, we reduced the data set to 90,637 detectable tryptic peptide sequences fitting the analytical windows of common high-resolution mass spectrometers.…”

How many spots with missing values can be tolerated in quantitative two-dimensional gel electrophoresis when applying univariate statistics?

“…All mass spectra for the peptide mass fingerprint (PMF) were recorded in positive ion reflectron mode with delayed extraction by accumulating up to 500 single unselected laser pulses and were internally calibrated using peptides from trypsin autolysis. The lists of monoisotopic m/z values for PMF searches were manually derived from unsmoothed mass spectra without baseline subtraction, omitting signals showing S/N ratios below 3:1, trypsin and keratin-related peptides [11], as well as matrix cluster ions [12]. Peak annotation was carried out automatically (Launchpad, Vers.…”