26 research outputs found
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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