Nonaqueous capillary electrophoresis in coated capillaries: an interesting alternative for proteomic applications |
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Authors: | Assunção Nilson A Deziderio Leandro A Grange Paulino Luis G Lupetti Karina O Carrilho Emanuel |
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Affiliation: | Instituto de Química de S?o Carlos, Universidade de S?o Paulo, S?o Carlos, Brazil. |
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Abstract: | This work brings together some contributions for the use of nonaqueous media for proteomic analysis, for both capillary electrophoresis (CE) separation and the preparation of tryptic digests. First, a ternary nonaqueous buffer consisting of 60/30/10 v/v methanol/acetonitrile/acetic acid with 12.5 mmol/L ammonium acetate was optimized for CE separation of the tryptic digest of lysozyme. Lysozyme was chosen as a model system for the protein digestion, which has also been prepared in an organic-rich medium with methanol/50 mmol/L NH(4)HCO(3), pH 8.0 (60/40 v/v). The separation results were compared to in silico (PeptideCutter program) digestion conditions, and high-efficiency peak separation (18 peaks) was obtained in 20 min with an electric field of 350 V/cm. In addition, we have evaluated the stability of a coated capillary with poly-N,N-dimethylacrylamide (60/30 cm total/effective length and 75 microm ID) for over 100 runs of tryptic digest with the nonaqueous background electrolyte solvent system. The migration times for ten selected peptide peaks presented 3-7% relative standard deviation. |
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Keywords: | Capillary coating Nonaqueous capillary electrophoresis Tryptic digests |
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