Sequencing of rat liver cytosolic proteins by matrix-assisted laser desorption ionization-quadrupole time-of-flight mass spectrometry following electrophoretic separation and extraction.
A new technique is described that enables the direct determination of the complete or partial amino acid sequence of cytosolic proteins separated by gel electrophoresis and allows for the further observation of disease- or drug-induced posttranslational modifications. The procedure uses a two-phase extraction strategy (ethyl acetate/ammonium bicarbonate) for the efficient separation of proteins/peptides from an electrophoretic matrix and subsequent sequence analysis by matrix-assisted laser desorption ionization-quadrupole time-of-flight mass spectrometry. The method was tested using hepatocyte cytosolic proteins and compared to a complementary approach using direct solvent extraction from in-gel digests. Although the latter procedure identified the proteins, it did not enable complete amino acid sequence determination. In contrast, high sequence coverage was obtained using the peptide extraction procedure, without any apparent dependence on protein size. The technique minimized the chemically inconsistent modifications generated from in-gel digestion, thus aiding mass spectrometric interpretation and valid protein sequence identification.
She
Yi-Min
YM
Department of Physics and Astronomy, Faculty of Science, University of Manitoba, Man, R3T 2N2, Winnipeg, Canada.
Wang
Gu-Qi
GQ
Loboda
Alexander
A
Ens
Werner
W
Standing
Kenneth G
KG
Burczynski
Frank J
FJ
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008099:Liver / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020539:Sequence Analysis, Protein / Q000379:methods*; D012697:Serine Endopeptidases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011506:Proteins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D016282:Aspartic Acid Endopeptidases