title: Studies of histidine as a suitable isoelectric buffer for tryptic digestion and isoelectric trapping fractionation followed by capillary electrophoresis-mass spectrometry for proteomic analysis.
abstract: The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using "membrane separated wells for isoelectric focusing and trapping" (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers. In addition, this sample preparation method and MSWIFT separations have been coupled with capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as an alternative separation approach for proteomic studies. Tryptic peptides of ribosomal proteins in histidine are fractionated using MSWIFT followed by CE-MALDI-MS, which further illustrates the ability to couple fractions from a pI based separation device to CE-MS. Specifically, two-dimensional CE-MS plots provide a direct correlation between the numbers of basic residues within the peptide sequence displayed in charge-state trend lines. Combining MSWIFT and CE-MS provides added information regarding peptide sequence, specifically pI and in-solution charge state. Post-translational modifications can also be identified using this method.
authors: Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
authors: Stephanie M
authors: SM
authors: Cologna
authors: Brad J
authors: BJ
authors: Williams
authors: William K
authors: WK
authors: Russell
authors: Pei-Jing
authors: PJ
authors: Pai
authors: Gyula
authors: G
authors: Vigh
authors: David H
authors: DH
authors: Russell
mesh_terms: D000818:Animals; D002021:Buffers; D002417:Cattle; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D019075:Electrophoresis, Capillary; D006639:Histidine / Q000737:chemistry*; D007525:Isoelectric Focusing; D010446:Peptide Fragments / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism*
chemical_list: D002021:Buffers; D010446:Peptide Fragments; D020543:Proteome; D012710:Serum Albumin, Bovine; D006639:Histidine; D014357:Trypsin