High-throughput Database Search and Large-scale Negative Polarity Liquid Chromatography–Tandem Mass Spectrometry with Ultraviolet Photodissociation for Complex Proteomic Samples

Abstract

The use of ultraviolet photodissociation (UVPD) for the activation and dissociation of peptide anions is evaluated for broader coverage of the proteome. To facilitate interpretation and assignment of the resulting UVPD mass spectra of peptide anions, the MassMatrix database search algorithm was modified to allow automated analysis of negative polarity MS/MS spectra. The new UVPD algorithms were developed based on the MassMatrix database search engine by adding specific fragmentation pathways for UVPD. The new UVPD fragmentation pathways in MassMatrix were rigorously and statistically optimized using two large data sets with high mass accuracy and high mass resolution for both MS 1 and MS 2 data acquired on an Orbitrap mass spectrometer for complex Halobacterium and HeLa proteome samples. Negative mode UVPD led to the identification of 3663 and 2350 peptides for the Halo and HeLa tryptic digests, respectively, corresponding to 655 and 645 peptides that were unique when compared with electron transfer dissociation (ETD), higher energy collision-induced dissociation, and collision-induced dissociation results for the same digests analyzed in the positive mode. In sum, 805 and 619 proteins were identified via UVPD for the Halobacterium and HeLa samples, respectively, with 49 and 50 unique proteins identified in contrast to the more conventional MS/MS methods. The algorithm also features automated charge determination for low mass accuracy data, precursor filtering (including intact charge-reduced peaks), and the ability to combine both positive and negative MS/MS spectra into a single search, and it is freely open to the public. The accuracy and specificity of the MassMatrix UVPD search algorithm was also assessed for low resolution, low mass accuracy data on a linear ion trap. Analysis of a known mixture of three mitogen-activated kinases yielded similar sequence coverage percentages for UVPD of peptide anions versus conventional collisioninduced dissociation of peptide cations, and when these methods were combined into a single search, an increase of up to 13% sequence coverage was observed for the kinases. The ability to sequence peptide anions and cations in alternating scans in the same chromatographic run was also demonstrated. Because ETD has a significant bias toward identifying highly basic peptides, negative UVPD was used to improve the identification of the more acidic peptides in conjunction with positive ETD for the more basic species. In this case, tryptic peptides from the cytosolic section of HeLa cells were analyzed by polarity switching nanoLC-MS/MS utilizing ETD for cation sequencing and UVPD for anion sequencing. Relative to searching using ETD alone, positive/negative polarity switching significantly improved sequence coverages across identified proteins, resulting in a 33% increase in unique peptide identifications and more than twice the number of peptide spectral matches. Molecular & Cellular Proteomics 12: 10.1074/mcp.O113.028258, 2604-2614, 2013.The advent of new high-performance tandem mas...

The Negative Mode Proteome with Activated Ion Negative Electron Transfer Dissociation (AI-NETD)

“…GluC, which cleaves C-terminal to glutamic acid (and at slower rates, aspartic acid (60)), enabled the identification of more than 4,200 unique peptides, a greater than 7-fold increase over our previous NETD results with the enzyme (24). These peptides mapped back to 857 proteins, which also outmatches the best results achieved to date for any protease in negative mode approaches (25). Chymotrypsin and AspN performed considerably well, too, illustrating the flexibility AI-NETD can offer for peptide anion characterization.…”

“…Negative mode approaches for the characterization of peptide anions offer a valuable dimension to proteomic analyses (21,24,25,66), especially as biologically relevant posttranslational modifications and other analytes that pose challenges to canonical positive mode techniques continue to emerge (7,53,(67)(68)(69)(70)(71)(72). Platforms for shotgun analysis of complex mixtures of peptide anions have been introduced, most notably using UVPD and NETD; however, these approaches have yet to provide considerable proteomic depth (fewer than ϳ800 total proteins identified in a given experiment), restricting the degree to which peptide anion characterization can benefit the proteomic community.…”

“…We previously employed ammonium formate solvents for high-pH separations, but charge state distributions favoring lowly charged precursors, degradation of silica-based packing materials, and low precursor flux were significant challenges with this system (24,28). Although others have used similar systems with success, we also took note of several studies utilizing piperidine buffers for high-pH chromatography (20,25,53,54). With this knowledge, we evaluated five different solvent systems using either piperidine or ammonium formate buffers over triplicate 90-min nLC-MS/MS analysis of yeast peptides with AI-NETD fragmentation.…”

“…Alterna-tively, a number of emerging fragmentation techniques, including electron-based dissociation methods and photodissociation approaches, can generate sequence informative MS/MS spectra from peptide anions (17)(18)(19)(20)(21)(22)(23). Both negative electron transfer dissociation (NETD) and ultraviolet photodissociation (UVPD) have been employed in large-scale proteomic studies, enabling sequencing of thousands of unique peptides in a single experiment (24,25). In NETD, the negative mode analog of electron transfer dissociation (ETD) (26), peptide anions are oxidized with reagent cations, causing the radical peptide anions to undergo electron rearrangement steps that often lead to cleavage of the C-C ␣ backbone bond, producing a…”

Cysteine-Selective Peptide Identification: Selenium-Based Chromophore for Selective S–Se Bond Cleavage with 266 nm Ultraviolet Photodissociation

“…7 Ultraviolet photodissociation (UVPD) is a more recent activation technique that has gained traction and has been applied to a broad range of proteomics applications. [8][9][10][11][12][13][14][15] In particular, UVPD using 193 nm photons generates very rich fragmentation patterns, 10,15 providing high sequence coverage of peptides.…”