Standardisation of rapid in-gel digestion by mass spectrometry |
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Authors: | Granvogl Bernhard Gruber Patrick Eichacker Lutz Andreas |
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Institution: | 1. Department für Biologie I, Ludwig‐Maximilians‐Universit?t, München, Germany;2. OMX‐GmbH, München, Germany;3. Department für Biologie I, Ludwig‐Maximilians‐Universit?t, München, GermanyDepartment für Biologie I, Ludwig‐Maximilians‐Universit?t, Menzinger Strasse 67, D‐80638 München, Germany Fax: +0049‐89‐17861‐209=== |
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Abstract: | In-gel digestion has been standardised using a poly(propylene) disposable. We designed a four-step rapid and simple in-gel digestion protocol which is carried out in one self-contained reaction tube avoiding keratin contamination. In order to quantify the efficiency of in-gel digestion, we developed a rapid on-column peptide acetylation protocol. Results show that trypsin in-gel uptake is increased and in-gel digestion is 90% complete within 15 min. We further show that spectrum quality, peptide yield and sequence coverage for mass spectrometric analysis are enhanced. We utilise 2-D PAGE separation of photosystem II from barley to demonstrate that the protocol facilitates identification of highly hydrophobic membrane proteins. |
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Keywords: | Acetylation In‐gel digestion Peptide recovery Quantification Trypsin |
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