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 ∼10% of the band, (ii) cyanogen bromide (CNBr) cleavage of another ∼10% of the band, and (iii) enzymatic (endoproteinase Lys-C) digestion of the remaining ∼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.