Nanoliter-volume protein enrichment, tryptic digestion, and partial separation based on isoelectric points by CE for MALDI mass spectral analysis.
Sequence-specific proteolysis is an important part of protein identification by MS. Digestion of protein is commonly performed in-solution, in sample vials with volumes ranging from milli- to microliters. When digestion is performed with a sample volume below 1 microL, handling of solution and potential sample loss via adsorption become significant issues. In this report, a proof of concept for the digestion of a small volume protein solution inside a capillary was demonstrated using a discontinuous buffer system previously studied (Nesbitt, C. A., et al. J. Chromatogr. A 2005, 1073, 175-180). Upon voltage application, a pH junction was created by the discontinuous buffer. Using myoglobin as an example, the protein molecules were enriched at the junction with an estimated volume of a few nanoliters. A protease, trypsin, was then introduced to myoglobin at the junction by coenrichment to induce in-capillary digestion. The voltage application was then suspended to provide the necessary time (2 h) for the proteolysis to proceed. When completed, voltage application was resumed, and the discontinuous buffer reconcentrated the peptides formed from digestion. Importantly, the refocused peptides appeared to roughly elute according to their pIs, resulting in a partial separation. Direct sample deposition from capillary was performed to facilitate mass spectral analysis by MALDI. The partial separation, according to pI, offered the potential benefits of MALDI MS signal enhancement and provided supplementary pI information for peptide identity assignment.
Nesbitt
Chandra A
CA
Department of Chemistry, The University of Western Ontario, London, Ontario, Canada.
Jurcic
Kristina
K
Yeung
Ken K-C
KK
D002021:Buffers; D005591:Chemical Fractionation / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D044085:Microfluidics; D009211:Myoglobin / Q000302:isolation & purification; D036103:Nanotechnology / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism*
D002021:Buffers; D009211:Myoglobin; D011506:Proteins; D014357:Trypsin