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Evaluation of the efficiency of in-gel digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Shevchenko, Andrej |
| Copyright Year | 2001 |
| Abstract | Visualization of proteins separated by one-dimensional or two-dimensional polyacrylamide gel electrophoresis, in-gel digestion of excised protein bands (spots), followed by identification of proteins by mass spectrometry underpin many proteome characterization strategies (reviewed in (1–4)). A multitude of protocols for staining of polyacrylamide gels (reviewed in (5)) and of enzymatic in-gel digestion of proteins (reviewed in (1, 6)) at the low picomole–femtomole level has been reported. A combination of protein visualization and in-gel digestion methods designed for proteomic application is usually evaluated according to two major criteria. First, the lowest limit of reliable visualization of protein spot (band) is determined (5, 7, 8). Second, the sequence coverage of MALDI peptide mass fingerprints acquired from in-gel digests of the visualized protein spots and the sensitivity of mass spectrometric detection are evaluated (8–10). However, the latter criteria are particularly difficult to apply. The intensity of peptide signals detected in complex mixtures by MALDI MS strongly depends on the employed sample handling and probe preparation routines. Furthermore, unavoidable micro-heterogeneity of MALDI probes and inevitable presence of the residual amount of dyes, salts, and detergents result in significant shot-to-shot variation of peptide and matrix signals, requires consistent tuning of the laser fluence (11) and, consequently, does not allow quantitative comparison of spectra acquired in the separate experiments. |
| Starting Page | 1700 |
| Ending Page | 1705 |
| Page Count | 6 |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | https://publications.mpi-cbg.de/Shevchenko_2001_252.pdf |
| PubMed reference number | 11554724v1 |
| Volume Number | 296 |
| Issue Number | 2 |
| Journal | Analytical biochemistry |
| Language | English |
| Access Restriction | Open |
| Subject Keyword | Bands Detergents Exanthema Femtomole Gel Electrophoresis (lab technique) Handling (Psychology) Imagery Mass Spectrometry Polyacrylamide Gel Electrophoresis Protein Digestion (research activity) Protocols documentation Salts Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Staining method mixture picomole (pM) polyacrylamide gels |
| Content Type | Text |
| Resource Type | Article |
