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A robust approach for the analysis of peptides in the low femtomole range by capillary electrophoresis‐tandem mass spectrometry
Author(s) -
Neusüß Christian,
Pelzing Matthias,
Macht Marcus
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200209)23:18<3149::aid-elps3149>3.0.co;2-8
Subject(s) - chemistry , chromatography , capillary electrophoresis , mass spectrometry , tandem mass spectrometry , analytical chemistry (journal) , capillary action , capillary electrophoresis–mass spectrometry , ion trap , electrospray ionization , materials science , composite material
A capillary electrophoresis‐tandem mass spectrometry (CE‐MS/MS) approach has been developed for routine application in proteomic studies. Robustness of the coupling is achieved by using a standard coaxial sheath‐flow sprayer. Thereby, greater stability than nanoelectrospray ionization‐mass spectrometry coupling of sheathless capillary electrophoresis or nanoliquid chromatography (nano‐LC) is achieved, resulting in stable operation for several weeks and unattended overnight sequences. The applied sheath flow is reduced to 1–2 νL/min in order to increase sensitivity. Standard peptides and those of digests of standard proteins and gel‐separated proteins can be detected in the low femtomole range (full scan and MS/MS). Detection limits are found to be as low as 500 amol. Low femtomole amounts are required for unequivocal identification by MS/MS experiments in the ion trap and subsequent database search. By applying a simple pH‐mediated stacking the concentration sensitivity can be lowered to some tens of fmol/νL (n M ), depending on capillary size. This sensitivity is close to published values for sheathless CE‐MS and nano‐LC‐MS, respectively (a comparison to reference values is presented). Moreover, with capillaries of about 50 cm in length separations in less than 10 min are possible resulting in a throughput of up to four analyses per hour. This is a factor of 4–12 times faster than nano‐LC separation, being the state‐of‐the‐art techniques for proteomic studies.