Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis Journal Article
| Authors: | Erdjument-Bromage, H.; Lui, M.; Lacomis, L.; Grewal, A.; Annan, R. S.; McNulty, D. E.; Carr, S. A.; Tempst, P. |
| Article Title: | Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis |
| Abstract: | Mass spectrometry occupies a central position in most current protein identification schemes. So-called 'mass fingerprinting' techniques rely on composite mass patterns of proteolytic fragments, or dissociation products thereof, to query databases. Keys to successful analysis of ever smaller amounts are sensitivity and complete spectral information, both of which depend for a large part on proper sample preparation. Clean-up and concentration of peptide mixtures over eppendorf gel loading tips filled with chromatographic media (i.e. 'micro-tips') are believed to be quite useful in this regard. We have studied quantitative and qualitative aspects of polypeptide extraction using these small manual devices. Optimization of sample volume and additives, micro-tip bed volume, and eluent composition and volume, all contribute to effective recovery (~65-70%, on average). Improper digest conditions can, in fact, lead to far bigger losses, suggesting the need for at least trace amounts of Zwittergent 3-16. Of particular interest is our finding that partial fractionation, obtained by two-step micro-tip elution, generally results in more and better signals during subsequent mass analysis. Thus, by using optimized micro-tips, in combination with adequate sample handling and instrumentation, direct mass spectrometric identification can be routinely and successfully done in any resource facility type setting. Copyright (C) 1998 Elsevier Science B.V. |
| Keywords: | quantitative assay; mass spectrometry; protein analysis; amino acid sequence; molecular sequence data; saccharomyces cerevisiae; peptide fragments; peptides; chromatography, high pressure liquid; technique; solvents; trypsin; endopeptidases; spectrometry, mass, matrix-assisted laser desorption-ionization; reversed phase high performance liquid chromatography; glucosephosphate dehydrogenase; formic acids; priority journal; article; microanalysis; sample preparation; micro-tip liquid chromatography |
| Journal Title: | Journal of Chromatography A |
| Volume: | 826 |
| Issue: | 2 |
| ISSN: | 0021-9673 |
| Publisher: | Elsevier B.V. |
| Date Published: | 1998-11-27 |
| Start Page: | 167 |
| End Page: | 181 |
| Language: | English |
| DOI: | 10.1016/s0021-9673(98)00705-5 |
| PUBMED: | 9871337 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 12 December 2016 -- Source: Scopus |
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