Potential of Negative-Ion-Mode Proteomics: An MS1-Only Approach.

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This study investigates protein identification efficiency in the negative ionization mode using the DirectMS1 method. DirectMS1 is an ultrafast data acquisition method based on accurate peptide mass measurements and predicted retention times. Our method achieves the highest rate of protein identification in the negative ion mode to date, identifying over 1000 proteins in a human cell line at a 1% false discovery rate. This is accomplished using a single-shot 10 min separation gradient, comparable to lengthy MS/MS-based analyses. Optimizing separation and experimental conditions was achieved by utilizing mobile buffers containing 2.5 mM imidazole and 3% isopropanol. The study emphasized the complementary nature of data obtained in positive and negative ion modes. Combining the results from all replicates in both polarities increased the number of identified proteins to 1774. Additionally, we analyzed the method's efficiency using different proteases for protein digestion. Among the four studied proteases (LysC, GluC, AspN, and trypsin), trypsin and LysC demonstrated the highest protein identification yield. This suggests that digestion procedures utilized in positive-mode proteomics can be effectively applied in the negative ion mode. 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