Peptide mass fingerprint sequence coverage from differently stained proteins on two‐dimensional electrophoresis patterns by matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS)

Abstract

Identification of proteins separated by two-dimensional electrophoresis (2-DE) is a necessary task to overcome the purely descriptive character of 2-DE and a prerequisite to the construction of 2-DE databases in proteome projects. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver-stained protein spots by peptide mass fingerprinting. Unfortunately, proteins are modified by the silver staining procedure, leading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still clearly below the sequence coverage starting with Coomassie Brilliant Blue (CBB) R-250-stained spots. Other factors additionally seem to modify proteins during silver staining. By decreasing the protein amount, the advantage of very sensitive detection on the gel is lost during identification, because the resulting low sequence coverage is not sufficient for secure identification. Low-quantity proteins can be identified better starting with CBB G-250 or Zn-imidazol-stained proteins. In contrast, for high-quantity CBB R-250-stained spots, a sequence coverage of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium-quantity spots after combination of tryptic digest with Asp-N- and Glu-C digest.

Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to μ LC-ESI tandem mass spectrometry

“…Obtaining more than 50% of sequence coverage from a tryptic digest of a 2D-gel separated protein by either PMM or LC-MS/MS is difficult [47]. This was also found in our study as shown in Figure 3b, but it was also shown that relatively small proteins (0 -40 kDa) have a greater chance of obtaining sequence coverage of ϳ50% than medium to large proteins.…”

“…In some cases, greater than 80% sequence coverage was observed. In order to increase the chance of identifying modified sites, at least 80% sequence coverage is desired [47]. Use of other proteases with different cleavage specificity in addition to trypsin is a good way of increasing sequence coverage [14,49,50].…”

“…Use of other proteases with different cleavage specificity in addition to trypsin is a good way of increasing sequence coverage [14,49,50]. Jungblut and coworkers reported that they could achieve up to 80% sequence coverage for medium-quantity spots by PMM by employing two additional proteases, Asp-N and Glu-C, in addition to trypsin [47]. Therefore, by employing other proteases in addition to trypsin and by combining the matched peptides from two complementary protein identification methods, PMM and LC-MS/MS, maximum sequence coverage from a gel spot could be obtained, increasing the probability of identifying a PTM.…”

Identification and purification of a soluble region of BubR1: A critical component of the mitotic checkpoint complex

“…Interesting SDS-PAGE spots were excised from the stained gels, and the gel spots were sequentially destained, reduced, alkylated, and hydrolyzed, using modiWed porcine trypsin (Promega, Southhampton, UK), as previously described [34,35]. The extracted in-gel digested peptides were then identiWed via peptide mass Wngerprinting using MALDI-TOF MS, and the identiWed peptides were subsequently sequenced via MALDI-TOF/TOF tandem mass spectrometry (MS/MS) (Applied Biosystems, USA) at the Seoul National University College of Medicine, in Seoul, Korea [36]. Database searches were carried out using the search program, MASCOT, developed by Matrix Science Ltd (access is available on http:// www.matrix.science.com), and the ExPASy Molecular Biology Server at SWISS-PROT (http://www.expasy.ch).…”

Pharmacoproteomics in drug development

“…A tryptic digest (eg, peptide mixture) can be analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. 14 The measured and optimized monisotopic mass data then are compared with theoretically derived peptide mass databases, generated by applying specific enzymatic cleavage rules to predicted/known protein sequences. High-throughput MAL-DI-based peptide mass fingerprinting (PMF) is accurate and sensitive.…”