Matrix-assisted laser-desorption/ionization mass spectrometric approaches for the identification of gel-separated proteins in the 5-50 pmol range.
The ability to identify and characterize low picomole quantities of gel-separated proteins has greatly benefited from recent advancements in mass spectrometric analysis methods, particularly peptide-mass search routines. We are investigating the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)) to gain as much mass information as possible from a single gel-separated protein species. This report details results obtained from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separations, under nonreducing conditions, of known quantities of three proteins, followed by blotting to Immobilon-CD. Three methods were used to obtain MALDI-MS data from a single blotted protein band: (i) direct MALDI-MS of approximately 10% of the band, (ii) cyanogen bromide (CNBr) cleavage of another approximately 10% of the band, and (iii) enzymatic (endoproteinase Lys-C) digestion of the remaining approximately 70-80% of the band followed by MALDI-MS. At the level of 50 pmol of protein loaded onto the gel, data was obtained from all three approaches. At levels down to 5 pmol of protein loaded onto the gel, MALDI-MS data was obtained from the latter two methods, CNBr and Lys-C digestions, but not direct MALDI-MS. Sufficient peptide masses were obtained from the 5 pmol loads to identify two of the three test proteins using four mass search programs. Only limited peptide mass data was obtained from fetuin, a sialylated glycoprotein with six disulfides and no methionines, but it was identified.
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002364:Caseins / Q000302:isolation & purification; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses; D008567:Membranes, Artificial; D008832:Microchemistry; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D000509:alpha-Fetoproteins / Q000302:isolation & purification
D002364:Caseins; D008567:Membranes, Artificial; D009211:Myoglobin; D011506:Proteins; D000509:alpha-Fetoproteins