Nonaqueous capillary electrophoresis in coated capillaries:: An interesting alternative for proteomic applications

被引:14
作者
Assunçao, NA
Deziderio, LAG
Paulino, LG
Lupetti, KO
Carrilho, E
机构
[1] Univ Sao Paulo, Inst Quim Sao Carlos, Lab Cromatog, BR-13560970 Sao Carlos, Brazil
[2] Univ Fed Sao Carlos, Dept Engn Prod Quim, BR-13560 Sao Carlos, SP, Brazil
关键词
capillary coating; nonaqueous capillary electrophoresis; tryptic digests;
D O I
10.1002/elps.200500195
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 NH4HCO3, pH 8.0 (60/40v/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 mu m 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.
引用
收藏
页码:3292 / 3299
页数:8
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