Here we report a novel approach in which gel-separated proteins are guanidinated in-gel prior to enzymatic cleavage. In contrast to previously described techniques, this procedure allows the extracted tryptic peptides to be N-terminal sulfonated without any further sample purification. The derivatized peptides were subsequently fragmented using a matrix-assisted laser desorption/ionization time of flight/time of flight instrument. The approach facilitates the de novo sequence analysis and allows obtaining longer stretches of amino acid sequence information. We demonstrate that the obtained information can be used to identify proteins using a sequence similarity search algorithm. The technique was compared to the standard peptide mass fingerprint approach, applied either in-gel or in solution, using a number of sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated model proteins. Finally, the new protocol was applied on a proteomic study of two-dimensional PAGE separated proteins from Shewanella oneidensis. More than 50 proteins from this organism were identified using sub-picomol quantities of protein, and peptide sequences of up to 20 amino acid residues in length have been determined.