Proteolytic (18)O labeling is a very powerful tool for differential analysis applied to proteome studies. However, it is a relatively new technique and the optimization of the labeling process still needs some attention. We found that the two-step post-proteolytic labeling should be favored over the conventional digestion of proteins in H(2) (18)O, since the former allows for higher sample concentrations and thus more favorable kinetics. It was demonstrated that the inhibitory effect of urea on (18)O incorporation could be compensated by the use of higher sample concentrations. Furthermore, it was shown that heat-deactivation of trypsin prevents (18)O/(16)O back-exchange. In addition, no non-specific hydrolysis of the peptides could be observed as a result of the heating. Heat inactivation of trypsin opens the way for the use of capillary electrophoresis as a separation technique in proteolytic labeling studies, as it abolishes the need for use of detrimental additives. Analysis of a labeled protein digest by capillary isoelectric focusing/mass spectrometry showed the applicability of the method. No back-exchange was observed across the entire electropherogram.
Peptide profiles of single neurons in Lymnaea stagnalis were directly characterized by matrix-assisted laser desorptiowionization mass spectrometry. The mass analysis was performed after minor pretreatment and without any separation steps. Good-quality spectra were obtained of several cell types and also other tissues. The results were compared with the results of conventional peptide chemical methods. In addition to many known peptides, several new peptides were identified. The method provides new opportunities for studying peptide compositions at the single-cell level, which is shown to have many advantages.
Significant improvements in the absolute detection limits for proteins in matrix-assisted laser desorptiod ionization mass spectrometry (MALDI-MS) are demonstrated by the application of so-called picolitre vials. By the reduction of the sample volume from a few pL down to 250 pL and simultaneous reduction of the sample spot area from a few mm2 down to 0.01 mm2, low attomole detection limits are obtained for bradykinin and cytochrome c. The detection limit in a single-shot mass spectrum of bradykinin is estimated to be as low as 250 zeptomol. These are currently the lowest amounts of protein and the smallest volumes analyzed by MALDI-MS.
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