Development of ultraviolet photodissociation for high-throughput analysis of heavily modified proteins and peptides

The utility of 193 nm ultraviolet photodissociation (UVPD) is evaluated for high-throughput proteomics applications including: analysis of small peptides in a traditional bottom-up proteomics workflow, analysis of heavily modified larger middle down sized peptides, and heavily modified intact proteins in a top-down proteomics workflow. UVPD uses higher energy ultraviolet photons (193 nm, 6.4 eV per photon), which are absorbed by the backbone to activate and dissociate ions effectively. UVPD dissociation is able to generate extensive backbone fragmentation enabling excellent characterization of peptides and proteins compared to traditional methods. Moreover, UVPD is also less hindered by certain experimental variables such as degree of modification, charge state and even ion polarity. These features are easily capitalized on for proteomics applications especially analysis of post translational modifications (PTM’s). Characterization of PTM’s is of great interest due to their involvement in several important cellular processes including cell signaling, tumorigenesis and gene expression. The studies covered in this work focus on utilizing the unique capabilities of UVPD to: 1.) characterize underrepresented peptides (acidic peptides and phosphopeptides) in the negative polarity including development of software for the analysis of the data generated, 2.) analyze intact proteins which have undergone extensive chemical modification and charge state augmentation, and 3.) precisely characterize histone proteins which are heavily modified due to their central role in gene expression and other transcription related functions.Chemistr

Top-Down Characterization of Heavily Modified Histones Using 193 nm Ultraviolet Photodissociation Mass Spectrometry

The characterization of protein post-translational modifications (PTMs) remains a significant challenge for traditional bottom-up proteomics methods owing to the lability of PTMs and the difficulty of mapping combinatorial patterns of PTMs based on analysis of small peptides. These shortcomings have accelerated interest in top-down MS/MS methods that focus on analysis of intact proteins. Simultaneous mapping of all PTMs requires extensive sequence coverage to confidently localize modifications. 193 nm ultraviolet photodissociation (UVPD) has been shown to generate unparalleled sequence coverage for intact proteins compared to traditional MS/MS methods. This study focuses on identification and localization of PTMs of histones by UVPD, higher-energy collisional dissociation (HCD), and the hybrid method electron-transfer/higher-energy collision dissociation (EThcD) via a high throughput liquid chromatography–mass spectrometry strategy. In total, over 500 proteoforms were characterized among these three activation methods with 46% of the identifications found in common by two or more activation methods. EThcD and UVPD afforded more extensive characterization of proteoforms than HCD with average gains in sequence coverage of 15% and <i>C</i>-scores that doubled on average

Integrating Carbamylation and Ultraviolet Photodissociation Mass Spectrometry for Middle-Down Proteomics

The most popular bottom-up proteomics workflow uses trypsin to enzymatically cleave proteins C-terminal to lysine and arginine residues prior to LCMS/MS analysis of the resulting peptides. The high frequency of these residues generates short peptides, some of which are too small or uninformative for optimal analysis and which potentially contribute to gaps in sequence coverage of proteins. Analysis of larger peptides, termed “middle-down”, has the potential to span greater sections of protein sequences if the larger peptides are adequately characterized based on their fragmentation patterns. We describe a strategy to generate larger peptides in conjunction with successful characterization by ultraviolet photodissociation (UVPD) for MS/MS analysis in a middle-down workflow, as demonstrated for proteins from <i>E. coli</i> lysates. The larger peptides are produced via modification of lysine residues by carbamylation of proteins. Carbamylation of proteins followed by tryptic digestion produced peptides similar to those expected from Arg-C proteolysis, yet with fewer missed and nonspecific cleavages. UVPD provides excellent sequence coverage of the larger peptides that are often less well characterized by traditional collision-based activation methods

Impact of Protease on Ultraviolet Photodissociation Mass Spectrometry for Bottom-up Proteomics

Recent mass spectrometric studies have reported enhanced proteome coverage by employing multiple proteases or by using multiple or alternative activation methods such as electron-transfer dissociation in combination with collisional-activated dissociation (CAD). In this study the use of 193 nm ultraviolet photodissociation for the analysis of thousands of <i>Halobacterium salinarum</i> peptides generated by four proteases (trypsin, LysC, GluC, and chymotrypsin) was evaluated in comparison with higher energy CAD (HCD). Proteins digested by trypsin resulted in greater sequence coverage for HCD over UVPD. LysC digestion resulted in similar sequence coverages for UVPD and HCD; however, for proteins digested by GluC and chymotrypsin 5–10% more sequence coverage on average was achieved by UVPD. HCD resulted in more peptide identifications (at 1% false discovery rate) for trypsin (4356 peptides by HCD versus 3907 peptides by UVPD), whereas UVPD identified greater numbers of peptides for LysC digests (1033 peptides by UVPD versus 844 HCD), chymotrypsin digests (3219 peptides for UVPD versus 2921 for HCD), and GluC digests (2834 peptides for UVPD and 2393 for HCD) and correspondingly greater numbers of proteins

Impact of Protease on Ultraviolet Photodissociation Mass Spectrometry for Bottom-up Proteomics

Recent mass spectrometric studies have reported enhanced proteome coverage by employing multiple proteases or by using multiple or alternative activation methods such as electron-transfer dissociation in combination with collisional-activated dissociation (CAD). In this study the use of 193 nm ultraviolet photodissociation for the analysis of thousands of <i>Halobacterium salinarum</i> peptides generated by four proteases (trypsin, LysC, GluC, and chymotrypsin) was evaluated in comparison with higher energy CAD (HCD). Proteins digested by trypsin resulted in greater sequence coverage for HCD over UVPD. LysC digestion resulted in similar sequence coverages for UVPD and HCD; however, for proteins digested by GluC and chymotrypsin 5–10% more sequence coverage on average was achieved by UVPD. HCD resulted in more peptide identifications (at 1% false discovery rate) for trypsin (4356 peptides by HCD versus 3907 peptides by UVPD), whereas UVPD identified greater numbers of peptides for LysC digests (1033 peptides by UVPD versus 844 HCD), chymotrypsin digests (3219 peptides for UVPD versus 2921 for HCD), and GluC digests (2834 peptides for UVPD and 2393 for HCD) and correspondingly greater numbers of proteins

Improvement of Shotgun Proteomics in the Negative Mode by Carbamylation of Peptides and Ultraviolet Photodissociation Mass Spectrometry

Although acidic peptides compose a substantial portion of many proteomes, their less efficient ionization during positive polarity electrospray ionization (ESI) impedes their detection in bottom-up mass spectrometry workflows. We have implemented a derivatization strategy based on carbamylation which converts basic amine sites (Lys, N-termini) to less basic amides for enhanced analysis in the negative mode. Ultraviolet photodissociation (UVPD) is used to analyze the resulting peptide anions, as demonstrated for tryptic peptides from bovine serum albumin and <i>Halobacterium salinarum</i> in a high throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) mode. LC/UVPD-MS of a carbamylated <i>H. salinarum</i> digest resulted in 45% more identified peptides and 25% more proteins compared to the unmodified digest analyzed in the negative mode

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

Orbitrap mass spectrometry and high-field asymmetric waveform ion mobility spectrometry (FAIMS) enable the in-depth analysis of human serum proteoforms

Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncation, single amino acid substitutions and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches which cannot recapitulate the original proteoform content of samples. Lengthy sample preparation and the need for extensive prefractionation to mitigate proteoform dynamic range issues are likely factors preventing clinical laboratories from routinely performing top-down experiments. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on depletion of albumin and immunoglobulins followed by simplified fractionation of remaining serum proteins via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS), which served as an additional separation dimension to further simplify serum proteoforms mixtures. This LC-FAIMS-MS2 method led to the identification of over 1,000 serum proteoforms <30 kDa using a reduced number of experiments, more than doubling the number of proteoforms identified in previous studies