13 research outputs found
Data-Independent Acquisition Proteomics and N‑Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts
Myocardial ischemia-reperfusion (IR) (stunning) injury
triggers
changes in the proteome and degradome of the heart. Here, we utilize
quantitative proteomics and comprehensive degradomics to investigate
the molecular mechanisms of IR injury in isolated rat hearts. The
control group underwent aerobic perfusion, while the IR injury group
underwent 20 min of ischemia and 30 min of reperfusion to induce a
stunning injury. As MMP-2 activation has been shown to contribute
to myocardial injury, hearts also underwent IR injury with ARP-100,
an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2
to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy,
we quantified 4468 proteins in ventricular extracts, whereby 447 proteins
showed significant alterations among the three groups. We then used
subtiligase-mediated N-terminomic labeling to identify more than a
hundred specific cleavage sites. Among these protease substrates,
15 were identified following IR injury. We identified alterations
in numerous proteins involved in mitochondrial function and metabolism
following IR injury. Our findings provide valuable insights into the
biochemical mechanisms of myocardial IR injury, suggesting alterations
in reactive oxygen/nitrogen species handling and generation, fatty
acid metabolism, mitochondrial function and metabolism, and cardiomyocyte
contraction
Data-Independent Acquisition Proteomics and N‑Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts
Myocardial ischemia-reperfusion (IR) (stunning) injury
triggers
changes in the proteome and degradome of the heart. Here, we utilize
quantitative proteomics and comprehensive degradomics to investigate
the molecular mechanisms of IR injury in isolated rat hearts. The
control group underwent aerobic perfusion, while the IR injury group
underwent 20 min of ischemia and 30 min of reperfusion to induce a
stunning injury. As MMP-2 activation has been shown to contribute
to myocardial injury, hearts also underwent IR injury with ARP-100,
an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2
to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy,
we quantified 4468 proteins in ventricular extracts, whereby 447 proteins
showed significant alterations among the three groups. We then used
subtiligase-mediated N-terminomic labeling to identify more than a
hundred specific cleavage sites. Among these protease substrates,
15 were identified following IR injury. We identified alterations
in numerous proteins involved in mitochondrial function and metabolism
following IR injury. Our findings provide valuable insights into the
biochemical mechanisms of myocardial IR injury, suggesting alterations
in reactive oxygen/nitrogen species handling and generation, fatty
acid metabolism, mitochondrial function and metabolism, and cardiomyocyte
contraction
N‑Terminomic Identification of Intracellular MMP‑2 Substrates in Cardiac Tissue
Proteases are enzymes
that induce irreversible post-translational
modifications by hydrolyzing amide bonds in proteins. One of these
proteases is matrix metalloproteinase-2 (MMP-2), which has been shown
to modulate extracellular matrix remodeling and intracellular proteolysis
during myocardial injury. However, the substrates of MMP-2 in heart
tissue are limited, and lesser known are the cleavage sites. Here,
we used degradomics to investigate the substrates of intracellular
MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively
active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified
from mammalian cells. Using this protease, we proteolyzed ventricular
extracts and used subtiligase-mediated N-terminomic labeling which
identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass
spectrometry. The intracellular MMP-2 cleavage sites identified in
heart tissue extracts were enriched for proteins primarily involved
in metabolism, as well as the breakdown of fatty acids and amino acids.
We further characterized the cleavage of three of these MMP-2-Fc substrates
based on the gene ontology analysis. We first characterized the cleavage
of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2
substrate in myocardial injury. We then characterized the cleavage
of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1),
representing new cardiac tissue substrates. Our findings provide insights
into the intracellular substrates of MMP-2 in cardiac cells, suggesting
that MMP-2 activation plays a role in cardiac metabolism
N‑Terminomic Identification of Intracellular MMP‑2 Substrates in Cardiac Tissue
Proteases are enzymes
that induce irreversible post-translational
modifications by hydrolyzing amide bonds in proteins. One of these
proteases is matrix metalloproteinase-2 (MMP-2), which has been shown
to modulate extracellular matrix remodeling and intracellular proteolysis
during myocardial injury. However, the substrates of MMP-2 in heart
tissue are limited, and lesser known are the cleavage sites. Here,
we used degradomics to investigate the substrates of intracellular
MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively
active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified
from mammalian cells. Using this protease, we proteolyzed ventricular
extracts and used subtiligase-mediated N-terminomic labeling which
identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass
spectrometry. The intracellular MMP-2 cleavage sites identified in
heart tissue extracts were enriched for proteins primarily involved
in metabolism, as well as the breakdown of fatty acids and amino acids.
We further characterized the cleavage of three of these MMP-2-Fc substrates
based on the gene ontology analysis. We first characterized the cleavage
of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2
substrate in myocardial injury. We then characterized the cleavage
of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1),
representing new cardiac tissue substrates. Our findings provide insights
into the intracellular substrates of MMP-2 in cardiac cells, suggesting
that MMP-2 activation plays a role in cardiac metabolism
Data-Independent Acquisition Proteomics and N‑Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts
Myocardial ischemia-reperfusion (IR) (stunning) injury
triggers
changes in the proteome and degradome of the heart. Here, we utilize
quantitative proteomics and comprehensive degradomics to investigate
the molecular mechanisms of IR injury in isolated rat hearts. The
control group underwent aerobic perfusion, while the IR injury group
underwent 20 min of ischemia and 30 min of reperfusion to induce a
stunning injury. As MMP-2 activation has been shown to contribute
to myocardial injury, hearts also underwent IR injury with ARP-100,
an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2
to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy,
we quantified 4468 proteins in ventricular extracts, whereby 447 proteins
showed significant alterations among the three groups. We then used
subtiligase-mediated N-terminomic labeling to identify more than a
hundred specific cleavage sites. Among these protease substrates,
15 were identified following IR injury. We identified alterations
in numerous proteins involved in mitochondrial function and metabolism
following IR injury. Our findings provide valuable insights into the
biochemical mechanisms of myocardial IR injury, suggesting alterations
in reactive oxygen/nitrogen species handling and generation, fatty
acid metabolism, mitochondrial function and metabolism, and cardiomyocyte
contraction
Data-Independent Acquisition Proteomics and N‑Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts
Myocardial ischemia-reperfusion (IR) (stunning) injury
triggers
changes in the proteome and degradome of the heart. Here, we utilize
quantitative proteomics and comprehensive degradomics to investigate
the molecular mechanisms of IR injury in isolated rat hearts. The
control group underwent aerobic perfusion, while the IR injury group
underwent 20 min of ischemia and 30 min of reperfusion to induce a
stunning injury. As MMP-2 activation has been shown to contribute
to myocardial injury, hearts also underwent IR injury with ARP-100,
an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2
to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy,
we quantified 4468 proteins in ventricular extracts, whereby 447 proteins
showed significant alterations among the three groups. We then used
subtiligase-mediated N-terminomic labeling to identify more than a
hundred specific cleavage sites. Among these protease substrates,
15 were identified following IR injury. We identified alterations
in numerous proteins involved in mitochondrial function and metabolism
following IR injury. Our findings provide valuable insights into the
biochemical mechanisms of myocardial IR injury, suggesting alterations
in reactive oxygen/nitrogen species handling and generation, fatty
acid metabolism, mitochondrial function and metabolism, and cardiomyocyte
contraction
Data-Independent Acquisition Proteomics and N‑Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts
Myocardial ischemia-reperfusion (IR) (stunning) injury
triggers
changes in the proteome and degradome of the heart. Here, we utilize
quantitative proteomics and comprehensive degradomics to investigate
the molecular mechanisms of IR injury in isolated rat hearts. The
control group underwent aerobic perfusion, while the IR injury group
underwent 20 min of ischemia and 30 min of reperfusion to induce a
stunning injury. As MMP-2 activation has been shown to contribute
to myocardial injury, hearts also underwent IR injury with ARP-100,
an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2
to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy,
we quantified 4468 proteins in ventricular extracts, whereby 447 proteins
showed significant alterations among the three groups. We then used
subtiligase-mediated N-terminomic labeling to identify more than a
hundred specific cleavage sites. Among these protease substrates,
15 were identified following IR injury. We identified alterations
in numerous proteins involved in mitochondrial function and metabolism
following IR injury. Our findings provide valuable insights into the
biochemical mechanisms of myocardial IR injury, suggesting alterations
in reactive oxygen/nitrogen species handling and generation, fatty
acid metabolism, mitochondrial function and metabolism, and cardiomyocyte
contraction
N‑Terminomic Identification of Intracellular MMP‑2 Substrates in Cardiac Tissue
Proteases are enzymes
that induce irreversible post-translational
modifications by hydrolyzing amide bonds in proteins. One of these
proteases is matrix metalloproteinase-2 (MMP-2), which has been shown
to modulate extracellular matrix remodeling and intracellular proteolysis
during myocardial injury. However, the substrates of MMP-2 in heart
tissue are limited, and lesser known are the cleavage sites. Here,
we used degradomics to investigate the substrates of intracellular
MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively
active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified
from mammalian cells. Using this protease, we proteolyzed ventricular
extracts and used subtiligase-mediated N-terminomic labeling which
identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass
spectrometry. The intracellular MMP-2 cleavage sites identified in
heart tissue extracts were enriched for proteins primarily involved
in metabolism, as well as the breakdown of fatty acids and amino acids.
We further characterized the cleavage of three of these MMP-2-Fc substrates
based on the gene ontology analysis. We first characterized the cleavage
of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2
substrate in myocardial injury. We then characterized the cleavage
of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1),
representing new cardiac tissue substrates. Our findings provide insights
into the intracellular substrates of MMP-2 in cardiac cells, suggesting
that MMP-2 activation plays a role in cardiac metabolism
N‑Terminomic Identification of Intracellular MMP‑2 Substrates in Cardiac Tissue
Proteases are enzymes
that induce irreversible post-translational
modifications by hydrolyzing amide bonds in proteins. One of these
proteases is matrix metalloproteinase-2 (MMP-2), which has been shown
to modulate extracellular matrix remodeling and intracellular proteolysis
during myocardial injury. However, the substrates of MMP-2 in heart
tissue are limited, and lesser known are the cleavage sites. Here,
we used degradomics to investigate the substrates of intracellular
MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively
active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified
from mammalian cells. Using this protease, we proteolyzed ventricular
extracts and used subtiligase-mediated N-terminomic labeling which
identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass
spectrometry. The intracellular MMP-2 cleavage sites identified in
heart tissue extracts were enriched for proteins primarily involved
in metabolism, as well as the breakdown of fatty acids and amino acids.
We further characterized the cleavage of three of these MMP-2-Fc substrates
based on the gene ontology analysis. We first characterized the cleavage
of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2
substrate in myocardial injury. We then characterized the cleavage
of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1),
representing new cardiac tissue substrates. Our findings provide insights
into the intracellular substrates of MMP-2 in cardiac cells, suggesting
that MMP-2 activation plays a role in cardiac metabolism
Data-Independent Acquisition Proteomics and N‑Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts
Myocardial ischemia-reperfusion (IR) (stunning) injury
triggers
changes in the proteome and degradome of the heart. Here, we utilize
quantitative proteomics and comprehensive degradomics to investigate
the molecular mechanisms of IR injury in isolated rat hearts. The
control group underwent aerobic perfusion, while the IR injury group
underwent 20 min of ischemia and 30 min of reperfusion to induce a
stunning injury. As MMP-2 activation has been shown to contribute
to myocardial injury, hearts also underwent IR injury with ARP-100,
an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2
to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy,
we quantified 4468 proteins in ventricular extracts, whereby 447 proteins
showed significant alterations among the three groups. We then used
subtiligase-mediated N-terminomic labeling to identify more than a
hundred specific cleavage sites. Among these protease substrates,
15 were identified following IR injury. We identified alterations
in numerous proteins involved in mitochondrial function and metabolism
following IR injury. Our findings provide valuable insights into the
biochemical mechanisms of myocardial IR injury, suggesting alterations
in reactive oxygen/nitrogen species handling and generation, fatty
acid metabolism, mitochondrial function and metabolism, and cardiomyocyte
contraction