8 research outputs found
<i>Staphylococcus aureus</i> Surface Proteins Involved in Adaptation to Oxacillin Identified Using a Novel Cell Shaving Approach
Staphylococcus aureus is a Gram-positive
pathogen responsible for a variety of infections, and some strains
are resistant to virtually all classes of antibiotics. Cell shaving
proteomics using a novel probability scoring algorithm to compare
the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted
to high levels of oxacillin (APT). APT displayed altered cell morphology
compared with COL and increased aggregation in biofilm assays. Increased
resistance to β-lactam antibiotics was observed, but adaptation
to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150
proteins at a threshold determined by the scoring algorithm. Proteins
unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR
and SACOL2302. Quantitative RT-PCR showed increased expression of <i>sacol2302</i> in APT grown with oxacillin (>6-fold compared
with COL). Overexpression of <i>sacol2302</i> in COL to
levels consistent with APT (+ oxacillin) did not influence biofilm
formation or β-lactam resistance. Proteomics using iTRAQ and
LC–MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated
elevated APT abundances of LCP proteins, capsule and peptidoglycan
biosynthesis proteins, and proteins involved in wall remodelling.
Adaptation to oxacillin also induced urease proteins, which maintained
culture pH compared to COL. These results show that S. aureus modifies surface architecture in response
to antibiotic adaptation
Large-Scale Capture of Peptides Containing Reversibly Oxidized Cysteines by Thiol-Disulfide Exchange Applied to the Myocardial Redox Proteome
Redox
regulation is emerging as an important post-translational
modification in cell signaling and pathogenesis. Cysteine (Cys) is
the most redox active of the commonly coded amino acids and is thus
an important target for redox-based modifications. Reactions that
oxidize the Cys sulfur atom to low oxidation states (e.g., disulfide)
are reversible, while further reactions to higher oxidation states
(e.g., sulfonic acid) may be irreversible under biological conditions.
Reversible modifications are particularly interesting as they mediate
redox signaling and regulation of proteins under physiological conditions
and during adaptation to oxidant stress. An enrichment method that
relied on rapid and specific alkylation of free Cys, followed by thiol-based
reduction and resin capture by thiol-disulfide exchange chemistry
was applied to isolate reversibly modified Cys-containing peptides.
Chromatographic conditions were optimized to provide increased specificity
by removal of noncovalent interactions. The technique was highly efficient,
based on near equimolar reactions with the resin, reproducible and
linear for peptide elution, as quantified by label-free mass spectrometry.
The method was applied to a complex protein lysate generated from
rat myocardial tissue and 6559 unique Cys-containing peptides from
2694 proteins were identified. Comparison with the rat database and
previous studies showed effective enrichment of proteins modified
by S-nitrosylation, disulfide formation, and Cys-sulfenic acid. Analysis
of amino acid sequence features indicated a preference for acidic
residues and increased hydrophilicity in the regions immediately up-
or downstream of the reactive Cys. This technique is ideally suited
for the enrichment and profiling of reversible Cys modifications on
a proteome-wide scale
Large-Scale Capture of Peptides Containing Reversibly Oxidized Cysteines by Thiol-Disulfide Exchange Applied to the Myocardial Redox Proteome
Redox
regulation is emerging as an important post-translational
modification in cell signaling and pathogenesis. Cysteine (Cys) is
the most redox active of the commonly coded amino acids and is thus
an important target for redox-based modifications. Reactions that
oxidize the Cys sulfur atom to low oxidation states (e.g., disulfide)
are reversible, while further reactions to higher oxidation states
(e.g., sulfonic acid) may be irreversible under biological conditions.
Reversible modifications are particularly interesting as they mediate
redox signaling and regulation of proteins under physiological conditions
and during adaptation to oxidant stress. An enrichment method that
relied on rapid and specific alkylation of free Cys, followed by thiol-based
reduction and resin capture by thiol-disulfide exchange chemistry
was applied to isolate reversibly modified Cys-containing peptides.
Chromatographic conditions were optimized to provide increased specificity
by removal of noncovalent interactions. The technique was highly efficient,
based on near equimolar reactions with the resin, reproducible and
linear for peptide elution, as quantified by label-free mass spectrometry.
The method was applied to a complex protein lysate generated from
rat myocardial tissue and 6559 unique Cys-containing peptides from
2694 proteins were identified. Comparison with the rat database and
previous studies showed effective enrichment of proteins modified
by S-nitrosylation, disulfide formation, and Cys-sulfenic acid. Analysis
of amino acid sequence features indicated a preference for acidic
residues and increased hydrophilicity in the regions immediately up-
or downstream of the reactive Cys. This technique is ideally suited
for the enrichment and profiling of reversible Cys modifications on
a proteome-wide scale
<i>Staphylococcus aureus</i> Surface Proteins Involved in Adaptation to Oxacillin Identified Using a Novel Cell Shaving Approach
Staphylococcus aureus is a Gram-positive
pathogen responsible for a variety of infections, and some strains
are resistant to virtually all classes of antibiotics. Cell shaving
proteomics using a novel probability scoring algorithm to compare
the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted
to high levels of oxacillin (APT). APT displayed altered cell morphology
compared with COL and increased aggregation in biofilm assays. Increased
resistance to β-lactam antibiotics was observed, but adaptation
to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150
proteins at a threshold determined by the scoring algorithm. Proteins
unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR
and SACOL2302. Quantitative RT-PCR showed increased expression of <i>sacol2302</i> in APT grown with oxacillin (>6-fold compared
with COL). Overexpression of <i>sacol2302</i> in COL to
levels consistent with APT (+ oxacillin) did not influence biofilm
formation or β-lactam resistance. Proteomics using iTRAQ and
LC–MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated
elevated APT abundances of LCP proteins, capsule and peptidoglycan
biosynthesis proteins, and proteins involved in wall remodelling.
Adaptation to oxacillin also induced urease proteins, which maintained
culture pH compared to COL. These results show that S. aureus modifies surface architecture in response
to antibiotic adaptation
Comparative Proteomics and Glycoproteomics Reveal Increased N‑Linked Glycosylation and Relaxed Sequon Specificity in <i>Campylobacter jejuni</i> NCTC11168 O
Campylobacter jejuni is a major
cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the
N-glycosylation of membrane-associated proteins. We examined two variants
of the genome sequenced strain NCTC11168: O, a representative of the
original clinical isolate, and GS, a laboratory-adapted relative of
O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography
coupled to tandem mass spectrometry (2D-LC–MS/MS) allowed the
confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at
statistically significant altered levels of abundance between variants.
Proteins associated with the O variant included adhesins (CadF and
FlpA), proteases, capsule biosynthesis, and cell shape determinants
as well as six proteins encoded by the Pgl system, including the PglK
flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted
specific glycoproteins more abundant in NCTC11168 O, whereas others
remained unaltered. Hydrophilic interaction liquid chromatography
(HILIC) and LC–MS/MS identified 30 completely novel glycosites
from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein
(Cj0455c) was identified that did not contain the <i>C. jejuni </i>N-linked sequon D/E-X-<u>N</u>-X-S/T (X ≠Pro) but that instead contained a
sequon with leucine at the −2 position. Occupied atypical sequons
were also observed in Cj0958c (OxaA; Gln at the −2 position)
and Cj0152c (Ala at the +2 position). The relative O and GS abundances
of 30 glycopeptides were determined by label-free quantitation, which
revealed a >100-fold increase in the atypical glycopeptide from
Cj0455c
in isolate O. Our data provide further evidence for the importance
of the Pgl system in <i>C. jejuni</i>
Release of Tissue-specific Proteins into Coronary Perfusate as a Model for Biomarker Discovery in Myocardial Ischemia/Reperfusion Injury
Diagnosis of acute coronary syndromes is based on protein
biomarkers,
such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB)
that are released into the circulation. Biomarker discovery is focused
on identifying very low abundance tissue-derived analytes from within
albumin-rich plasma, in which the wide dynamic range of the native
protein complement hinders classical proteomic investigations. We
employed an <i>ex vivo</i> rabbit model of myocardial ischemia/reperfusion
(I/R) injury using Langendorff buffer perfusion. Nonrecirculating
perfusate was collected over a temporal profile of 60 min reperfusion
following brief, reversible ischemia (15 min; 15I/60R) for comparison
with irreversible I/R (60I/60R). Perfusate proteins were separated
using two-dimensional gel electrophoresis (2-DE) and identified by
mass spectrometry (MS), revealing 26 tissue-specific proteins released
during reperfusion post-15I. Proteins released during irreversible
I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional
gel electrophoresis coupled to liquid chromatography and tandem mass
spectrometry; geLC–MS) and gel-free (LC–MS/MS) methods.
A total of 192 tissue-specific proteins were identified during reperfusion
post-60I. Identified proteins included those previously associated
with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples
currently under investigation in large cohort studies (heart-type
fatty acid binding protein; FABPH). The postischemic release profile
of a novel cardiac-specific protein, cysteine and glycine-rich protein
3 (Csrp3; cardiac LIM domain protein) was validated by Western blot
analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion
following acute myocardial infarction. These studies indicate that
animal modeling of biomarker release using <i>ex vivo</i> buffer perfused tissue to limit the presence of obfuscating plasma
proteins may identify candidates for further study in humans
Release of Tissue-specific Proteins into Coronary Perfusate as a Model for Biomarker Discovery in Myocardial Ischemia/Reperfusion Injury
Diagnosis of acute coronary syndromes is based on protein
biomarkers,
such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB)
that are released into the circulation. Biomarker discovery is focused
on identifying very low abundance tissue-derived analytes from within
albumin-rich plasma, in which the wide dynamic range of the native
protein complement hinders classical proteomic investigations. We
employed an <i>ex vivo</i> rabbit model of myocardial ischemia/reperfusion
(I/R) injury using Langendorff buffer perfusion. Nonrecirculating
perfusate was collected over a temporal profile of 60 min reperfusion
following brief, reversible ischemia (15 min; 15I/60R) for comparison
with irreversible I/R (60I/60R). Perfusate proteins were separated
using two-dimensional gel electrophoresis (2-DE) and identified by
mass spectrometry (MS), revealing 26 tissue-specific proteins released
during reperfusion post-15I. Proteins released during irreversible
I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional
gel electrophoresis coupled to liquid chromatography and tandem mass
spectrometry; geLC–MS) and gel-free (LC–MS/MS) methods.
A total of 192 tissue-specific proteins were identified during reperfusion
post-60I. Identified proteins included those previously associated
with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples
currently under investigation in large cohort studies (heart-type
fatty acid binding protein; FABPH). The postischemic release profile
of a novel cardiac-specific protein, cysteine and glycine-rich protein
3 (Csrp3; cardiac LIM domain protein) was validated by Western blot
analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion
following acute myocardial infarction. These studies indicate that
animal modeling of biomarker release using <i>ex vivo</i> buffer perfused tissue to limit the presence of obfuscating plasma
proteins may identify candidates for further study in humans
Release of Tissue-specific Proteins into Coronary Perfusate as a Model for Biomarker Discovery in Myocardial Ischemia/Reperfusion Injury
Diagnosis of acute coronary syndromes is based on protein
biomarkers,
such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB)
that are released into the circulation. Biomarker discovery is focused
on identifying very low abundance tissue-derived analytes from within
albumin-rich plasma, in which the wide dynamic range of the native
protein complement hinders classical proteomic investigations. We
employed an <i>ex vivo</i> rabbit model of myocardial ischemia/reperfusion
(I/R) injury using Langendorff buffer perfusion. Nonrecirculating
perfusate was collected over a temporal profile of 60 min reperfusion
following brief, reversible ischemia (15 min; 15I/60R) for comparison
with irreversible I/R (60I/60R). Perfusate proteins were separated
using two-dimensional gel electrophoresis (2-DE) and identified by
mass spectrometry (MS), revealing 26 tissue-specific proteins released
during reperfusion post-15I. Proteins released during irreversible
I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional
gel electrophoresis coupled to liquid chromatography and tandem mass
spectrometry; geLC–MS) and gel-free (LC–MS/MS) methods.
A total of 192 tissue-specific proteins were identified during reperfusion
post-60I. Identified proteins included those previously associated
with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples
currently under investigation in large cohort studies (heart-type
fatty acid binding protein; FABPH). The postischemic release profile
of a novel cardiac-specific protein, cysteine and glycine-rich protein
3 (Csrp3; cardiac LIM domain protein) was validated by Western blot
analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion
following acute myocardial infarction. These studies indicate that
animal modeling of biomarker release using <i>ex vivo</i> buffer perfused tissue to limit the presence of obfuscating plasma
proteins may identify candidates for further study in humans