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

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.

Evaluation of capillaries with different inner coatings for DNA analysis using dilute polymer solutions by capillary electrophoresis

“…25 As an alternative capillary coating, we used another covalently bound coating of PDMA, a polymer with hydrophobic characteristics. 26 Based on successful results acquired with uncoated capillaries, described by Barron et al, 1 we also used this kind of capillary in our experiments for comparison.…”

“…The PDMA coated capillary was 50 µm i.d. and 27 cm in length (20 cm effective length and was coated with PDMA in house 26 by adaptation of an earlier published method. 34 Basically, new capillaries were cleaned with acetone (10 min), 1 mol L -1 NaOH (30 min), 0.1 mol L -1 HCl (10 min), and finally washed with water (10 min).…”

“…The PDMA capillary was manufactured in our laboratory 26 according to a previously published procedure 34 and, to the best of our knowledge, this is the first application of this coating and HEC polymer solutions for the separation of DNA molecules. The HEC concentrations selected for this study were 0.15, 0.25, 0.50, 1.00, 1.50 and 1.80% because this range of concentration has shown good results for separations in the PVA capillary.…”

Eletroforese capilar acoplada à espectrometria de massas (CE-MS): vinte anos de desenvolvimento

“…Alguns exemplos de desenvolvimentos nesta direção, começando pela instrumentação, são o acoplamento de microchips ou MS, desenvolvimento de colunas especiais para CE-MS sheathless, microcolunas empacotadas para eletrocromatografia e métodos eletroforéticos em meio não-aquoso. 28 Particularmente na área de proteômica, o foco é voltado para a proteômica clínica, buscando biomarcadores para diagnóstico precoce de doenças e no entendimento de como algumas doenças evoluem, como esquizofrenia e Alzheimer. O interesse em metabólitos secundários abrange desde a detecção e identificação estrutural de novos metabólitos biologicamente ativos, 122,129 até o entendimento de rotas metabólicas e mecanismo de ação de proteínas numa abordagem mais ampla de química biológica e biologia de sistemas.…”

“…Uma das vantagens que estes tipos de eletrólitos oferecem é a formação de um spray estável e compatível com o sistema MS. 26,27 Este fenômeno ocorre principalmente ao se utilizar capilares com revestimento polimérico interno e eletrólitos contendo modificadores orgânicos (como formamida, metanol e acetonitrila), em análises de proteínas e peptídeos. [28][29][30] Outros modos de separação que empregam eletrólitos mais elaborados, como isotacoforese capilar transiente (t-CITP -transient capillary isotacophoresis), focalização isoelétrica capilar (CIEFcapillary isoeletric focusing) e cromatografia eletrocinética micelar (MEKC -micellar electrokinetic chromatography), também podem ser utilizados. [31][32][33][34] Nestes casos, para evitar a contaminação do detector pelos componentes não voláteis do eletrólito (tensoativos, ciclodextrinas, etc), uma alternativa de acoplamento denominada preenchimento parcial (PF -partial filling) foi proposta.…”