A robust approach for the analysis of peptides in the low femtomole range by capillary electrophoresis-tandem mass spectrometry.
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 microL/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/microL (nM), 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.
Neusüss
Christian
C
Bruker Saxonia Analytik GmbH, Leipzig, Germany. cne@bsax.de
Pelzing
Matthias
M
Macht
Marcus
M
D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods* / Q000706:statistics & numerical data; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods / Q000706:statistics & numerical data; D014168:Transferrin / Q000032:analysis
D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D012709:Serum Albumin; D014168:Transferrin