On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis.
Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300-1800. Good focusing was achieved for insulin receptor, which produced ~10 s peak width. For 0.1 mg mL(-1) bovine serum albumin (BSA) digests, 24±1 peptides (sequence coverage 47±4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
Guijie
G
Zhu
Liangliang
L
Sun
Ping
P
Yang
Norman J
NJ
Dovichi
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D002460:Cell Line; D019075:Electrophoresis, Capillary; D007525:Isoelectric Focusing; D051379:Mice; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D010455:Peptides; D011506:Proteins; D012710:Serum Albumin, Bovine; D014357:Trypsin