The field of proteomics almost uniformly relies on peptide cation analysis, leading to an underrepresentation of acidic portions of proteomes, including relevant acidic posttranslational modifications. Despite the many benefits negative mode proteomics can offer, peptide anion analysis remains in its infancy due mainly to challenges with high-pH reversed-phase separations and a lack of robust fragmentation methods suitable for peptide anion characterization. Here, we report the first implementation of activated ion negative electron transfer dissociation (AI-NETD) on the chromatographic timescale, generating 7,601 unique peptide identifications from Saccharomyces cerevisiae in single-shot nLC-MS/MS analyses of tryptic peptides-a greater than 5-fold increase over previous results with NETD alone. These improvements translate to identification of 1,106 proteins, making this work the first negative mode study to identify more than 1,000 proteins in any system. We then compare the performance of AI-NETD for analysis of peptides generated by five proteases (trypsin, LysC, GluC, chymotrypsin, and AspN) for negative mode analyses, identifying as many as 5 Global protein analysis continues to enjoy substantial technological leaps forward in its ability to characterize protein expression in a variety of organisms with both speed and sensitivity (1-4). Nevertheless, the impressive advances in protein sequence technology over past decades have rigidly adhered to positive electrospray ionization for MS 1 analysis, limiting the scope of peptides and posttranslational modifications that can be analyzed. Widely utilized acidic mobile phases both permit stable and reproducible reversed-phase separations and also provide optimal conditions for ionization and detection of peptides and proteins that readily accept positive charge via protonation (i.e. basic species). Acidic peptides and proteins, however, favor deprotonation, making positive electrospray regimes ill suited for their characterization. Moreover, important classes of posttranslational modifications (PTMs), such as phosphorylation, sulfation, and glycosylation, can impart acidic properties to the peptides and proteins they modify, often producing entire classes of biomolecules that preferentially ionize as anions (5-10).Electrospray ionization operated in the negative mode can generate multiply deprotonated species (11, 12); however, canonical collisional activation methods produce MS/MS spectra riddled with neutral losses and internal fragments that are difficult, if not impossible, to interpret (13-16). AlternaFrom the ‡Department