Nonaqueous capillary electrophoresis in coated capillaries: an interesting alternative for proteomic applications.
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.
Assunção
Nilson A
NA
Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, Brazil.
Deziderio
Leandro A Grange
LA
Paulino
Luis G
LG
Lupetti
Karina O
KO
Carrilho
Emanuel
E
D002021:Buffers; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D040901:Proteomics; D015203:Reproducibility of Results; D014357:Trypsin / Q000737:chemistry
D002021:Buffers; D014357:Trypsin