Effect of ionization mode in the analysis of proteolytic protein digests

The bottom-up sequencing approach relies on an accurate mass fingerprint of the peptide fragments produced from enzymatic digestion. However, this technique is complicated by "dirty" mass spectra due to matrix interferences and, especially with regard to electrospray ionization data, the presence of multiply-charged species, both of which produce false and incorrect hits in database searches. Here, it is shown that the quality of mass spectra of peptide digests can be improved significantly by changing the polarity of the mass spectrometer's ionization source. Using the negative ion mode in both matrix-assisted laser desorption ionization and electrospray ionization of peptide digests results in the detection of a number of peaks in the mass spectra that are not observed in positive ion mode. In addition, there is considerable reduction in interference from contaminant species in the negative ion mode versus the positive ion mode. By combining the positive ion and negative ion mode data, greater sequence coverage and improved database search scores were obtained. When digestion is incomplete, a dramatic enrichment in the observed abundances for digest product peptides versus the intact proteins could be obtained by changing the pH and the ionization mode. The present results highlight the importance of using positive ion mode and negative ion mode combined to improve overall amino acid sequence coverage and improve confidence in protein identification.