The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer’s disease and in cancer pathogenesis. The 5 nanosecond, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision-based and electron-based dissociation methods.

Top-down proteomics is the analysis of intact proteins in their endogenous form without proteolysis, preserving valuable information about post-translation modifications, isoforms and proteolytic processing. The quality of top-down LC-MS/MS datasets is rapidly increasing due to advances in instrumentation and sample processing protocols. However, the top-down mass spectra are substantially more complex compared to conventional bottom-up data. To take full advantage of the increasing data quality, there is an urgent need to develop algorithms and software tools for confident proteoform identification and quantification. In this study, we present a new open source software suite for top-down proteomics analysis consisting of an LC-MS feature finding algorithm, a database search algorithm, and an interactive results viewer. The presented tool along with several other popular tools were evaluated using human-in-mouse xenograft luminal and basal breast tumor samples that are known to have significant differences in protein abundance based on bottom-up analysis.

Intact protein characterization using mass spectrometry thus far has been achieved at the cost of throughput. Presented here is the application of 193 nm ultraviolet photodissociation (UVPD) for top down identification and characterization of proteins in complex mixtures in an online fashion. Liquid chromatographic separation at the intact protein level coupled with fast UVPD and high-resolution detection resulted in confident identification of 46 unique sequences compared to 44 using HCD from prepared Escherichia coli ribosomes. Importantly, nearly all proteins identified in both the UVPD and optimized HCD analyses demonstrated a substantial increase in confidence in identification (as defined by an average decrease in E value of ∼40 orders of magnitude) due to the higher number of matched fragment ions. Also shown is the potential for high-throughput characterization of intact proteins via liquid chromatography (LC)–UVPD-MS of molecular weight-based fractions of a Saccharomyces cerevisiae lysate. In total, protein products from 215 genes were identified and found in 292 distinct proteoforms, 168 of which contained some type of post-translational modification.

We provide the initial performance evaluation of a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer operating at the Environmental Molecular Sciences Laboratory at the Pacific Northwest National Laboratory. The spectrometer constructed for the 21T system employs a commercial dual linear ion trap mass spectrometer coupled to a FTICR spectrometer designed and built in-house. Performance gains from moving to higher magnetic field strength are exemplified by the measurement of peptide isotopic fine structure, complex natural organic matter mixtures, and large proteins. Accurate determination of isotopic fine structure was demonstrated for doubly charged Substance P with minimal spectral averaging, and 8158 molecular formulas assigned to Suwannee River Fulvic Acid standard with root-mean-square (RMS) error of 10 ppb. We also demonstrated superior performance for intact proteins; namely, broadband isotopic resolution of the entire charge state distribution of apo-transferrin (78 kDa) and facile isotopic resolution of monoclonal antibody under a variety of acquisition parameters (e.g., 6 s time-domains with absorption mode processing yielded resolution of approximately 1 M at m/z = 2700). Graphical Abstract ᅟ.

Secondary organic aerosol (SOA), formed in the photooxidation of diesel fuel, biodiesel fuel, and 20% biodiesel fuel/80% diesel fuel mixture, are prepared under high-NO conditions in the presence and absence of sulfur dioxide (SO), ammonia (NH), and relative humidity (RH). The composition of condensed-phase organic compounds in SOA is measured using several complementary techniques including aerosol mass spectrometry (AMS), high-resolution nanospray desorption electrospray ionization mass spectrometry (nano-DESI/HRMS), and ultrahigh resolution and mass accuracy 21T Fourier transform ion cyclotron resonance mass spectrometry (21T FT-ICR MS). Results demonstrate that sulfuric acid and condensed organosulfur species formed in photooxidation experiments with SO are present in the SOA particles. Fewer organosulfur species are formed in the high humidity experiments, performed at RH 90%, in comparison with experiments done under dry conditions. There is a strong overlap of organosulfur species observed in this study with previous field and chamber studies of SOA. Many MS peaks of organosulfates (R-OS(O)OH) previously designated as biogenic or of unknown origin in field studies might have originated from anthropogenic sources, such as photooxidation of hydrocarbons present in diesel and biodiesel fuel.

Compared to traditional collision induced dissociation methods, electron capture dissociation (ECD) provides more comprehensive characterization of large peptides and proteins as well as preserves labile post-translational modifications. However, ECD experiments are generally restricted to the high magnetic fields of FTICR-MS that enable the reaction of large polycations and electrons. Here, we demonstrate the use of an electromagnetostatic ECD cell to perform ECD and hybrid ECD methods utilizing 193 nm photons (ECuvPD) or collisional activation (EChcD) in a benchtop quadrupole-Orbitrap mass spectrometer. The electromagnetostatic ECD cell was designed to replace the transfer octapole between the quadrupole and C-trap. This implementation enabled facile installation of the ECD cell, and ions could be independently subjected to ECD, UVPD, HCD, or any combination. Initial benchmarking and characterization of fragmentation propensities for ECD, ECuvPD, and EChcD were performed using ubiquitin (8.6 kDa). ECD yielded extensive sequence coverage for low charge states of ubiquitin as well as for the larger protein carbonic anhydrase II (29 kDa), indicating pseudo-activated ion conditions. Additionally, relatively high numbers of d- and w-ions enable differentiation of isobaric isoleucine and leucine residues and suggest a distribution of electron energies yield hot-ECD type fragmentation. We report the most comprehensive characterization to date for model proteins up to 29 kDa and a monoclonal antibody at the subunit level. ECD, ECuvPD, and EChcD yielded 93, 95, and 91% sequence coverage, respectively, for carbonic anhydrase II (29 kDa), and targeted online analyses of monoclonal antibody subunits yielded 86% overall antibody sequence coverage.

Pilot Project #1—the identification and characterization of human histone H4 proteoforms by top-down MS—is the first project launched by the Consortium for Top-Down Proteomics (CTDP) to refine and validate top-down MS. Within the initial results from seven participating laboratories, all reported the probability-based identification of human histone H4 (UniProt accession P62805) with expectation values ranging from 10−13 to 10−105. Regarding characterization, a total of 74 proteoforms were reported, with 21 done so unambiguously; one new PTM, K79ac, was identified. Inter-laboratory comparison reveals aspects of the results that are consistent, such as the localization of individual PTMs and binary combinations, while other aspects are more variable, such as the accurate characterization of low-abundance proteoforms harboring >2 PTMs. An open-access tool and discussion of proteoform scoring are included, along with a description of general challenges that lie ahead including improved proteoform separations prior to mass spectrometric analysis, better instrumentation performance, and software development.

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...