Two-dimensional gel electrophoresis of any biological system presently resolves a plethora of highly purified proteins for which no function or identity has been determined. Theoretical and experimental data were used to demonstrate that peptide-mass fingerprinting (PMF) could aid in the recognition of conserved motifs across species boundaries, and thereby assist in attributing putative function to some of these molecules. Amino acids residue substitutions produced by biological diversity and phylogenetic distance combine to highlight regions of functional significance within proteins. Using 10 prokaryotic and two eukaryotic elongation factors (EF), up to 25 peptide fragments (> 800 Da) per molecule were compared across species boundaries within a 12 x 12 contingency table (66 cross-species comparisons), based upon the degree of molecular mass and amino acid sequence identity. Total amino acid sequence identity ranged from 29.4-80.9% for these molecules. Peptide fragments with homologous sequence across three or more EF were defined as containing, or being near to, conserved functional motifs. Twelve such fragments (> 800 Da) were found in this group of proteins. In addition, an 808.9 Da peptide of unknown functional significance was seen to occur in three of the 12 molecules studied and in another three EF-Tu molecules. At the 83% (five of six residues) identity level, this fragment was found in a further 35 EF-Tu molecules and in 14 unrelated proteins. Further investigation should reveal a role for this fragment (motif) in structural integrity or protein function. A FASTA search conducted on a peptide fragment containing a conserved GTP-binding motif (GHVDHGK) of EF-Tu from Euglena gracilis was used as an example to putatively attribute partial function to three hypothetical proteins derived from DNA sequencing initiatives.