RESEARCH HIGHLIGHTS

PROTEOMICS Navigating the negative-mode proteome

A negative-mode electron-transfer dissociation (NETD) uses coverage ­compared with using trypsin alone. approach characterizes intractable parts electron-driven fragmentation to enable They detected 1,359 proteins (and more than of the proteome. sequencing of peptides ionized under nega- 21,000 peptides) by AI-NETD. Last, they The impressive advances in the tive electrospray conditions. applied extensive low-pH reversed-phase field in the past two decades were spurred in In recent work, Joshua Coon of the fractionation prior to AI-NETD. This pro- no small part by electrospray ionization, a University of Wisconsin-Madison and col- vided a major boost, yielding 3,730 protein gentle technique used to produce gas-phase leagues implemented activated ion negative identifications, representing 83% of the peptide ions for electron-transfer dissociation (AI-NETD) on expressed yeast proteome, with an average (MS/MS) analysis. The vast majority of stud- a large scale for the first time. In AI-NETD, sequence coverage of 44%. ies have relied on adding a positive charge peptide ions are irradiated with infrared light The Coon team’s AI-NETD peptide to ionize peptides. However, acidic peptides concurrently with the NETD reaction, which identifications overlapped by just 18% with will not readily accept a positive charge, improves peptide fragmentation over that those from a state-of-the-art positive-mode making them difficult to ionize. Many post-­ achieved with NETD alone. analysis, and 272 proteins were detected translationally modified peptides are acidic, Working in yeast, Coon and colleagues only with AI-NETD. AI-NETD may find its making large classes of modified peptides first tested just how much proteome cov- place as a powerful complement to current resistant to characterization. erage they could achieve in a single-shot proteomics approaches. Applying a negative charge to acidic pep- AI-NETD experiment using a single pro- Allison Doerr tides also results in their ionization, but this tease, trypsin, to break up the proteins into approach produces MS/MS spectra that are peptides. They detected 7,601 unique pep- RESEARCH PAPERS Riley, N.M. et al. The negative mode proteome with too complex to interpret using traditional tides from 1,106 proteins. Next, they tested activated ion negative electron transfer dissociation. fragmentation methods. However, there a combination of five different proteases, Mol. Cell. Proteomics doi:10.1074/mcp.M115.049726 are work-arounds. In particular, negative which has been shown to improve proteomic (20 July 2015). Nature America, Inc. All rights reserved. America, Inc. © 201 5 Nature npg

808 | VOL.12 NO.9 | SEPTEMBER 2015 | NATURE METHODS