Project description:Triplicate results for 193 nm UVPD, HCD, and EThcD of a mixture of 157 synthetic peptide mixture, and 193 nm UVPD, HCD, and EThcD of BB7.2 and W6/32 immunoprecipitations
Project description:The analysis of intact proteins (top-down strategy) by mass spectrometry has great potential to elucidate proteoform variation, including patterns of post-translational modifications (PTMs), which may not be discernible by analysis of peptides alone (bottom-up approach). To maximize sequence coverage and localization of PTMs, various fragmentation modes have been developed to produce fragment ions from deep within intact proteins. Ultraviolet photodissociation (UVPD) has recently been shown to produce high sequence coverage and PTM retention on a variety of proteins, with increasing evidence of efficacy on a chromatographic time scale. However, utilization of UVPD for high-throughput top-down analysis to date has been limited by bioinformatics. Here we detected 153 proteins and 489 proteoforms using UVPD and 271 proteins and 982 proteoforms using higher energy collisional dissociation (HCD) in a comparative analysis of HeLa whole-cell lysate by qualitative top-down proteomics. Of the total detected proteoforms, 286 overlapped between the UVPD and HCD data sets, with 68% of proteoforms having C scores greater than 40 for UVPD and 63% for HCD. The average sequence coverage (28 ± 20% for UVPD versus 17 ± 8% for HCD, p < 0.0001) was found to be higher for UVPD than HCD and with a trend toward improvement in q value for the UVPD data set. This study demonstrates the complementarity of UVPD and HCD for more extensive protein profiling and proteoform characterization.
Project description:Analysis of fragmentation under standard and supercharging conditions for intact proteins using HCD, ETD, EThcD, 213 nm UVPD, 193 nm UVPD
Project description:UVPD was implemented on an Orbitrap Q-Exactive plus equipped with a ESI/EP-MALDI. UVPD of MALDI generated ions was benchmarked against MALDI-ISD, MALDI-HCD and ESI-UVPD. MALDI UVPD outperformed MALDI HCD and ISD efficiently sequencing small proteins ions.
Project description:Application of 193 nm ultraviolet photodissociation (UVPD) for phosphoproteomics analysis of IMAC enriched HeLa and HCC70 lysates. HCC70 cells were either maintained in full media or serum starved for 6 hours prior to lysis. Each sample was analyzed by three UVPD and three HCD technical replicates using an Orbitrap Fusion mass spectrometer equipped with a Coherent 193 nm excimer laser. Using UVPD, a/x, b/y, and c/z ions were generated and improved phosphate retention was observed on product ions.
Project description:Middle Down UVPD MS of purified HeLa Histones analyzed using 193 nm Ultra violet photo dissocaiton on a Thermo Fisher Scientific Orbitrap Fusion Lumos
Project description:We evaluated 193 nm UVPD for characterization of TMT labeled peptides (HeLa digest) and N-glycopeptides (alpha-1-acid glycoprotein digest) at different conditions, and compared the results with HCD. Samples were digested with trypsin and labeled with TMT6. Instrument data files were searched with either MS-GF+ (using PNNL's DMS processing pipeline) or with Byonic.
Project description:Fragmentation trends of large peptides were characterized by five activation methods, including HCD, ETD, EThcD, 213 nm UVPD and 193 nm UVPD. Sequence coverages and scores were assessed based on charge site, peptide sequence and peptide size. Results from four model peptides, neuromedin, glucagon, galanin and amyloid B, showed a charge state dependence on sequence coverage for collision and electron-based activation methods. The effect of charge state and peptide size on sequence coverage was also explored for a Glu-C digest of E. coli ribosomal proteins, resulting in a maximum sequence coverage between 2-6 kDa depending on the activation method.
Project description:We analyzed the backbone fragmentation behavior of tryptic peptides of a four protein mixture and of E. coli lysate subjected to Ultraviolet Photodissociation (UVPD) at 213 nm on a commercially available UVPD-equipped tribrid mass spectrometer. We obtained 15,178 high-confidence peptide-spectrum matches by additionally recording a reference beam-type collision-induced dissociation (HCD) spectrum of each precursor. Type a, b and y ions were most prominent in UVPD spectra and median sequence coverage ranged from 5.8% (at 5 ms laser excitation time) to 45.0% (at 100 ms). Overall sequence fragment intensity remained relatively low (median: 0.4% (5 ms) to 16.8% (100 ms) of total intensity) and remaining precursor intensity high. Sequence coverage and sequence fragment intensity ratio correlated with precursor charge density, suggesting that UVPD at 213 nm may suffer from newly formed fragments sticking together due to non-covalent interactions. UVPD fragmentation efficiency therefore might benefit from supplemental activation, as was shown for ETD. Aromatic amino acids, most prominently tryptophan, facilitated UVPD. This points at aromatic tags as possible enhancers of UVPD. Data are available via ProteomeXchange and on spectrumviewer.org/db/UVPD_213nm_trypPep
Project description:In this study we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. 213 nm UVPD was compared to the commonly employed higher-energy collisional dissociation (HCD).