Theoretical prediction of the feasibility of identifing membrane proteins by MALDI-TOF

Carolina Garcia-Rizo1, Cristian Klein2, Pfeiffer, Siedler, Oesterhelt, Max-Planck Institute Biochemistry;, Max-Planck Institute Biochemistry

In the set of proteins of Halobacterium salinarum identified by MALDI-TOF peptide fingerprints, membrane proteins are severely underrepresented. The identification of protein by this technique is based on the matching of experimental and theoretical peptide masses in the typical MALDI-TOF measurement range [800-4000Da] . The possibility and reliability of identification increases with an increase in the number of matching peptides. The detection ratio of these tryptic peptides is mass dependent as is shown by a statistical analysis of measurements that resulted in reliable protein identification. This ratio is high in the range [1100-2900Da] (72% detection ratio), lower for small masses (45%) and much lower for high masses (34%). Further statistical study of theoretical digestions of all proteins from Halobacterium salinarum shows that membrane proteins have a lower number of tryptic peptides in the measurement range, and a higher fraction of them in the mass range >3000Da which has a low detection ratio. The required number of matching peptides for a reliable identification of a protein depends on the stringency applied. As we use high stringency in order to minimise false positives, higher scores and thus, higher numbers of matching peptides are needed. An analysis of the relationship between the number of matching peptides and its corresponding MASCOT score from all the measurements allows us to determine the number of peptides needed for a reliable identification. This minimal number is not reached for membrane proteins. Other statistical study is made using different proteases, chemical cleavage agents, or combinations of these, similar results were obtained. We can conclude therefore, that not enough peptides are obtained for a reliable identification by fingerprint analysis of membrane proteins in Halobacterium salinarum. Statistics for other organism were also performed to discriminate between organism specific and general effects. This method, can therefor be applied to any organism as a pre-test to evaluate the possible identification of its (membrane) proteins by mass fingerprints.