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Autofocusing and ESI‐MS analysis of protein digests in a miniaturized multicompartment electrolyzer
Author(s) -
Tomáš Roman,
Yan Liushui,
Křenková Jana,
Foret František
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200600802
Subject(s) - chemistry , chromatography , electrospray , mass spectrometry , fractionation , electrolysis , analytical chemistry (journal) , electrospray ionization , electrode , electrolyte
Free‐solution IEF of protein digests was studied in a newly introduced MicroRotofor™ multicompartment electrolyzer. The fractionation was performed in a cylindrical separation chamber divided into ten compartments with or without the addition of carrier ampholytes. In the case of autofocusing mode of operation, the tryptic digest itself served as the mixture of ampholytes leading to the separation of the peptides with well‐defined p I 's. The focusing process was monitored visually using colored p I markers. The resulting fractions from both modes of the separation were analyzed by CE and electrospray‐TOF mass spectrometer using electrospray tips microfabricated in polyimide. Additional experiments, aiming at visualization of the mass flux within the focusing compartments were performed using isotachophoretic migration of color cationic tracers. The study considered the autofocusing of both the peptides with well‐defined narrow p I 's as well as those showing negligible net charge in a broader pH range. Although not all peptides in the protein digests have well‐defined p I 's the autofocusing process can preseparate many of them leading to higher S/N in the ESI‐MS signals and improved protein sequence coverage.