Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database Searching of Positive and Negative Polarity 193 nm Ultraviolet Photodissociation Mass Spectra

To extend proteome coverage obtained from bottom-up mass spectrometry approaches, three complementary ion activation methods, higher energy collision dissociation (HCD), ultraviolet photodissociation (UVPD), and negative mode UVPD (NUVPD), are used to interrogate the tryptic peptides in a human hepatocyte lysate using a high performance Orbitrap mass spectrometer. The utility of combining results from multiple activation techniques (HCD+UVPD+NUVPD) is analyzed for total depth and breadth of proteome coverage. This study also benchmarks a new version of the Byonic algorithm, which has been customized for database searches of UVPD and NUVPD data. Searches utilizing the customized algorithm resulted in over 50% more peptide identifications for UVPD and NUVPD tryptic peptide data sets compared to other search algorithms. Inclusion of UVPD and NUVPD spectra resulted in over 600 additional protein identifications relative to HCD alone

Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database Searching of Positive and Negative Polarity 193 nm Ultraviolet Photodissociation Mass Spectra

To extend proteome coverage obtained from bottom-up mass spectrometry approaches, three complementary ion activation methods, higher energy collision dissociation (HCD), ultraviolet photodissociation (UVPD), and negative mode UVPD (NUVPD), are used to interrogate the tryptic peptides in a human hepatocyte lysate using a high performance Orbitrap mass spectrometer. The utility of combining results from multiple activation techniques (HCD+UVPD+NUVPD) is analyzed for total depth and breadth of proteome coverage. This study also benchmarks a new version of the Byonic algorithm, which has been customized for database searches of UVPD and NUVPD data. Searches utilizing the customized algorithm resulted in over 50% more peptide identifications for UVPD and NUVPD tryptic peptide data sets compared to other search algorithms. Inclusion of UVPD and NUVPD spectra resulted in over 600 additional protein identifications relative to HCD alone

Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database Searching of Positive and Negative Polarity 193 nm Ultraviolet Photodissociation Mass Spectra

To extend proteome coverage obtained from bottom-up mass spectrometry approaches, three complementary ion activation methods, higher energy collision dissociation (HCD), ultraviolet photodissociation (UVPD), and negative mode UVPD (NUVPD), are used to interrogate the tryptic peptides in a human hepatocyte lysate using a high performance Orbitrap mass spectrometer. The utility of combining results from multiple activation techniques (HCD+UVPD+NUVPD) is analyzed for total depth and breadth of proteome coverage. This study also benchmarks a new version of the Byonic algorithm, which has been customized for database searches of UVPD and NUVPD data. Searches utilizing the customized algorithm resulted in over 50% more peptide identifications for UVPD and NUVPD tryptic peptide data sets compared to other search algorithms. Inclusion of UVPD and NUVPD spectra resulted in over 600 additional protein identifications relative to HCD alone

Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database Searching of Positive and Negative Polarity 193 nm Ultraviolet Photodissociation Mass Spectra

To extend proteome coverage obtained from bottom-up mass spectrometry approaches, three complementary ion activation methods, higher energy collision dissociation (HCD), ultraviolet photodissociation (UVPD), and negative mode UVPD (NUVPD), are used to interrogate the tryptic peptides in a human hepatocyte lysate using a high performance Orbitrap mass spectrometer. The utility of combining results from multiple activation techniques (HCD+UVPD+NUVPD) is analyzed for total depth and breadth of proteome coverage. This study also benchmarks a new version of the Byonic algorithm, which has been customized for database searches of UVPD and NUVPD data. Searches utilizing the customized algorithm resulted in over 50% more peptide identifications for UVPD and NUVPD tryptic peptide data sets compared to other search algorithms. Inclusion of UVPD and NUVPD spectra resulted in over 600 additional protein identifications relative to HCD alone

Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database Searching of Positive and Negative Polarity 193 nm Ultraviolet Photodissociation Mass Spectra

To extend proteome coverage obtained from bottom-up mass spectrometry approaches, three complementary ion activation methods, higher energy collision dissociation (HCD), ultraviolet photodissociation (UVPD), and negative mode UVPD (NUVPD), are used to interrogate the tryptic peptides in a human hepatocyte lysate using a high performance Orbitrap mass spectrometer. The utility of combining results from multiple activation techniques (HCD+UVPD+NUVPD) is analyzed for total depth and breadth of proteome coverage. This study also benchmarks a new version of the Byonic algorithm, which has been customized for database searches of UVPD and NUVPD data. Searches utilizing the customized algorithm resulted in over 50% more peptide identifications for UVPD and NUVPD tryptic peptide data sets compared to other search algorithms. Inclusion of UVPD and NUVPD spectra resulted in over 600 additional protein identifications relative to HCD alone

Full-Featured Search Algorithm for Negative Electron-Transfer Dissociation

Negative electron-transfer dissociation (NETD) has emerged as a premier tool for peptide anion analysis, offering access to acidic post-translational modifications and regions of the proteome that are intractable with traditional positive-mode approaches. Whole-proteome scale characterization is now possible with NETD, but proper informatic tools are needed to capitalize on advances in instrumentation. Currently only one database search algorithm (OMSSA) can process NETD data. Here we implement NETD search capabilities into the Byonic platform to improve the sensitivity of negative-mode data analyses, and we benchmark these improvements using 90 min LC–MS/MS analyses of tryptic peptides from human embryonic stem cells. With this new algorithm for searching NETD data, we improved the number of successfully identified spectra by as much as 80% and identified 8665 unique peptides, 24 639 peptide spectral matches, and 1338 proteins in activated-ion NETD analyses, more than doubling identifications from previous negative-mode characterizations of the human proteome. Furthermore, we reanalyzed our recently published large-scale, multienzyme negative-mode yeast proteome data, improving peptide and peptide spectral match identifications and considerably increasing protein sequence coverage. In all, we show that new informatics tools, in combination with recent advances in data acquisition, can significantly improve proteome characterization in negative-mode approaches

Comparative Informatics Analysis to Evaluate Site-Specific Protein Oxidation in Multidimensional LC–MS/MS Data

Redox proteomics has yielded molecular insight into diseases of protein dysfunction attributable to oxidative stress, underscoring the need for robust detection of protein oxidation products. Additionally, oxidative protein surface mapping techniques utilize hydroxyl radicals to gain structural insight about solvent exposure. Interpretation of tandem mass spectral data is a critical challenge for such investigations, because reactive oxygen species target a wide breadth of amino acids. Additionally, oxidized peptides may be generated in a wide range of abundances since the reactivity of hydroxyl radicals with different amino acids spans 3 orders of magnitude. Taken together, these attributes of oxidative footprinting pose both experimental and computational challenges to detecting oxidized peptides that are naturally less abundant than their unoxidized counterparts. In this study, model proteins were oxidized electrochemically and analyzed at both the intact protein and peptide levels. A multidimensional chromatographic strategy was utilized to expand the dynamic range of oxidized peptide measurements. Peptide mass spectral data were searched by the “hybrid” software packages Inspect and Byonic, which incorporate <i>de novo</i> elements of spectral interpretation into a database search. This dynamic search capacity accommodates the challenge of searching for more than 40 oxidative mass shifts that can occur in a staggering variety of possible combinatorial occurrences. A prevailing set of oxidized residues was identified with this comparative approach, and evaluation of these sites was informed by solvent accessible surface area gleaned through molecular dynamics simulations. Along with increased levels of oxidation around highly reactive “hotspot” sites as expected, the enhanced sensitivity of these measurements uncovered a surprising level of oxidation on less reactive residues

Comparative Informatics Analysis to Evaluate Site-Specific Protein Oxidation in Multidimensional LC–MS/MS Data

Redox proteomics has yielded molecular insight into diseases of protein dysfunction attributable to oxidative stress, underscoring the need for robust detection of protein oxidation products. Additionally, oxidative protein surface mapping techniques utilize hydroxyl radicals to gain structural insight about solvent exposure. Interpretation of tandem mass spectral data is a critical challenge for such investigations, because reactive oxygen species target a wide breadth of amino acids. Additionally, oxidized peptides may be generated in a wide range of abundances since the reactivity of hydroxyl radicals with different amino acids spans 3 orders of magnitude. Taken together, these attributes of oxidative footprinting pose both experimental and computational challenges to detecting oxidized peptides that are naturally less abundant than their unoxidized counterparts. In this study, model proteins were oxidized electrochemically and analyzed at both the intact protein and peptide levels. A multidimensional chromatographic strategy was utilized to expand the dynamic range of oxidized peptide measurements. Peptide mass spectral data were searched by the “hybrid” software packages Inspect and Byonic, which incorporate <i>de novo</i> elements of spectral interpretation into a database search. This dynamic search capacity accommodates the challenge of searching for more than 40 oxidative mass shifts that can occur in a staggering variety of possible combinatorial occurrences. A prevailing set of oxidized residues was identified with this comparative approach, and evaluation of these sites was informed by solvent accessible surface area gleaned through molecular dynamics simulations. Along with increased levels of oxidation around highly reactive “hotspot” sites as expected, the enhanced sensitivity of these measurements uncovered a surprising level of oxidation on less reactive residues

Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry for Top-Down Characterization of <i>Pyrococcus furiosus</i> Proteins on a Proteome Scale

Intact protein analysis via top-down mass spectrometry (MS) provides the unique capability of fully characterizing protein isoforms and combinatorial post-translational modifications (PTMs) compared to the bottom-up MS approach. Front-end protein separation poses a challenge for analyzing complex mixtures of intact proteins on a proteomic scale. Here we applied capillary electrophoresis (CE) through a sheathless capillary electrophoresis-electrospray ionization (CESI) interface coupled to an Orbitrap Elite mass spectrometer to profile the proteome from <i>Pyrococcus furiosus</i>. CESI-top-down MS analysis of <i>Pyrococcus furiosus</i> cell lysate identified 134 proteins and 291 proteoforms with a total sample consumption of 270 ng in 120 min of total analysis time. Truncations and various PTMs were detected, including acetylation, disulfide bonds, oxidation, glycosylation, and hypusine. This is the largest scale analysis of intact proteins by CE-top-down MS to date

Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry for Top-Down Characterization of <i>Pyrococcus furiosus</i> Proteins on a Proteome Scale

Intact protein analysis via top-down mass spectrometry (MS) provides the unique capability of fully characterizing protein isoforms and combinatorial post-translational modifications (PTMs) compared to the bottom-up MS approach. Front-end protein separation poses a challenge for analyzing complex mixtures of intact proteins on a proteomic scale. Here we applied capillary electrophoresis (CE) through a sheathless capillary electrophoresis-electrospray ionization (CESI) interface coupled to an Orbitrap Elite mass spectrometer to profile the proteome from <i>Pyrococcus furiosus</i>. CESI-top-down MS analysis of <i>Pyrococcus furiosus</i> cell lysate identified 134 proteins and 291 proteoforms with a total sample consumption of 270 ng in 120 min of total analysis time. Truncations and various PTMs were detected, including acetylation, disulfide bonds, oxidation, glycosylation, and hypusine. This is the largest scale analysis of intact proteins by CE-top-down MS to date