Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis.
We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer. The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis.
Hess
D
D
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Covey
T C
TC
Winz
R
R
Brownsey
R W
RW
Aebersold
R
R
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D014357:Trypsin
D007527:Isoenzymes; D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin; D002256:Carbonic Anhydrases; D000103:Acetyl-CoA Carboxylase