75 research outputs found
Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human Fetuin
Fetuin,
also known as alpha-2-Heremans Schmid glycoprotein (AHSG),
belongs to some of the most abundant glycoproteins secreted into the
bloodstream. In blood, fetuins exhibit functions as carriers of metals
and small molecules. Bovine fetuin, which harbors 3 N-glycosylation
sites and a suggested half dozen O-glycosylation sites, has been used
often as a model glycoprotein to test novel analytical workflows in
glycoproteomics. Here we characterize and compare fetuin in depth,
using protein from three different biological sources: human serum,
bovine serum, and recombinant human fetuin expressed in HEK-293 cells,
with the aim to elucidate similarities and differences between these
proteins and the post-translational modifications they harbor. Combining
data from high-resolution native mass spectrometry and glycopeptide
centric LC-MS analysis, we qualitatively and quantitatively gather
information on fetuin protein maturation, N-glycosylation, O-glycosylation,
and phosphorylation. We provide direct experimental evidence that
both the human serum and part of the recombinant proteins are processed
into two chains (A and B) connected by a single interchain disulfide
bridge, whereas bovine fetuin remains a single-chain protein. Although
two N-glycosylation sites, one O-glycosylation site, and a phosphorylation
site are conserved from bovine to human, the stoichiometry of the
modifications and the specific glycoforms they harbor are quite distinct.
Comparing serum and recombinant human fetuin, we observe that the
serum protein harbors a much simpler proteoform profile, indicating
that the recombinant protein is not ideally engineered to mimic human
serum fetuin. Comparing the proteoform profile and post-translational
modifications of human and bovine serum fetuin, we observe that, although
the gene structures of these two proteins are alike, they represent
quite distinct proteins when their glycoproteoform profile is also
taken into consideration
Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human Fetuin
Fetuin,
also known as alpha-2-Heremans Schmid glycoprotein (AHSG),
belongs to some of the most abundant glycoproteins secreted into the
bloodstream. In blood, fetuins exhibit functions as carriers of metals
and small molecules. Bovine fetuin, which harbors 3 N-glycosylation
sites and a suggested half dozen O-glycosylation sites, has been used
often as a model glycoprotein to test novel analytical workflows in
glycoproteomics. Here we characterize and compare fetuin in depth,
using protein from three different biological sources: human serum,
bovine serum, and recombinant human fetuin expressed in HEK-293 cells,
with the aim to elucidate similarities and differences between these
proteins and the post-translational modifications they harbor. Combining
data from high-resolution native mass spectrometry and glycopeptide
centric LC-MS analysis, we qualitatively and quantitatively gather
information on fetuin protein maturation, N-glycosylation, O-glycosylation,
and phosphorylation. We provide direct experimental evidence that
both the human serum and part of the recombinant proteins are processed
into two chains (A and B) connected by a single interchain disulfide
bridge, whereas bovine fetuin remains a single-chain protein. Although
two N-glycosylation sites, one O-glycosylation site, and a phosphorylation
site are conserved from bovine to human, the stoichiometry of the
modifications and the specific glycoforms they harbor are quite distinct.
Comparing serum and recombinant human fetuin, we observe that the
serum protein harbors a much simpler proteoform profile, indicating
that the recombinant protein is not ideally engineered to mimic human
serum fetuin. Comparing the proteoform profile and post-translational
modifications of human and bovine serum fetuin, we observe that, although
the gene structures of these two proteins are alike, they represent
quite distinct proteins when their glycoproteoform profile is also
taken into consideration
Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human Fetuin
Fetuin,
also known as alpha-2-Heremans Schmid glycoprotein (AHSG),
belongs to some of the most abundant glycoproteins secreted into the
bloodstream. In blood, fetuins exhibit functions as carriers of metals
and small molecules. Bovine fetuin, which harbors 3 N-glycosylation
sites and a suggested half dozen O-glycosylation sites, has been used
often as a model glycoprotein to test novel analytical workflows in
glycoproteomics. Here we characterize and compare fetuin in depth,
using protein from three different biological sources: human serum,
bovine serum, and recombinant human fetuin expressed in HEK-293 cells,
with the aim to elucidate similarities and differences between these
proteins and the post-translational modifications they harbor. Combining
data from high-resolution native mass spectrometry and glycopeptide
centric LC-MS analysis, we qualitatively and quantitatively gather
information on fetuin protein maturation, N-glycosylation, O-glycosylation,
and phosphorylation. We provide direct experimental evidence that
both the human serum and part of the recombinant proteins are processed
into two chains (A and B) connected by a single interchain disulfide
bridge, whereas bovine fetuin remains a single-chain protein. Although
two N-glycosylation sites, one O-glycosylation site, and a phosphorylation
site are conserved from bovine to human, the stoichiometry of the
modifications and the specific glycoforms they harbor are quite distinct.
Comparing serum and recombinant human fetuin, we observe that the
serum protein harbors a much simpler proteoform profile, indicating
that the recombinant protein is not ideally engineered to mimic human
serum fetuin. Comparing the proteoform profile and post-translational
modifications of human and bovine serum fetuin, we observe that, although
the gene structures of these two proteins are alike, they represent
quite distinct proteins when their glycoproteoform profile is also
taken into consideration
Identification of Enriched PTM Crosstalk Motifs from Large-Scale Experimental Data Sets
Post-translational modifications
(PTMs) play an important role
in the regulation of protein function. Mass spectrometry based proteomics
experiments nowadays identify tens of thousands of PTMs in a single
experiment. A wealth of data has therefore become publically available.
Evidently the biological function of each PTM is the key question
to be addressed; however, such analyses focus primarily on single
PTM events. This ignores the fact that PTMs may act in concert in
the regulation of protein function, a process termed PTM crosstalk.
Relatively little is known on the frequency and functional relevance
of crosstalk between PTM sites. In a bioinformatics approach, we extracted
PTMs occurring in proximity in the protein sequence from publically
available databases. These PTMs and their flanking sequences were
subjected to stringent motif searches, including a scoring for evolutionary
conservation. Our unprejudiced approach was able to detect a respectable
set of motifs, of which about half were described previously. Among
these we could add many new proteins harboring these motifs. We extracted
also several novel motifs, which through their widespread appearance
and high conservation may pinpoint at previously nonannotated concerted
PTM actions. By employing network analyses on these proteins, we propose
putative functional roles for these novel motifs with two PTM sites
in close proximity
Identification of Enriched PTM Crosstalk Motifs from Large-Scale Experimental Data Sets
Post-translational modifications
(PTMs) play an important role
in the regulation of protein function. Mass spectrometry based proteomics
experiments nowadays identify tens of thousands of PTMs in a single
experiment. A wealth of data has therefore become publically available.
Evidently the biological function of each PTM is the key question
to be addressed; however, such analyses focus primarily on single
PTM events. This ignores the fact that PTMs may act in concert in
the regulation of protein function, a process termed PTM crosstalk.
Relatively little is known on the frequency and functional relevance
of crosstalk between PTM sites. In a bioinformatics approach, we extracted
PTMs occurring in proximity in the protein sequence from publically
available databases. These PTMs and their flanking sequences were
subjected to stringent motif searches, including a scoring for evolutionary
conservation. Our unprejudiced approach was able to detect a respectable
set of motifs, of which about half were described previously. Among
these we could add many new proteins harboring these motifs. We extracted
also several novel motifs, which through their widespread appearance
and high conservation may pinpoint at previously nonannotated concerted
PTM actions. By employing network analyses on these proteins, we propose
putative functional roles for these novel motifs with two PTM sites
in close proximity
Targeted Quantitative Phosphoproteomics Approach for the Detection of Phospho-tyrosine Signaling in Plants
Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal systems. However, a few studies have also reported Tyr phosphorylation in plants, but the relative contribution of tyrosine phosphorylation to plant signal transduction has remained an open question. We present an approach to selectively measure and quantify Tyr phosphorylation in plant cells, which can also be applied to whole plants. We combined a <sup>15</sup>N stable isotope metabolic labeling strategy with an immuno-affinity purification using phospho-tyrosine (pY) specific antibodies. This single enrichment strategy was sufficient to reproducibly identify and quantify pY containing peptides from total plant cell extract in a single LC–MS/MS run. We succeeded in identifying 149 unique pY peptides originating from 135 proteins, including a large set of different protein kinases and several receptor-like kinases. We used flagellin perception by <i>Arabidopsis</i> cells, a model system for pathogen triggered immune (PTI) signaling, to test our approach. We reproducibly quantified 23 pY peptides in 2 inversely labeled biological replicates identifying 11 differentially phosphorylated proteins. These include a set of 3 well-characterized flagellin responsive MAP kinases and 4 novel MAP kinases. With this targeted approach, we elucidate a new level of complexity in flagellin-induced MAP kinase activation
Targeted Quantitative Phosphoproteomics Approach for the Detection of Phospho-tyrosine Signaling in Plants
Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal systems. However, a few studies have also reported Tyr phosphorylation in plants, but the relative contribution of tyrosine phosphorylation to plant signal transduction has remained an open question. We present an approach to selectively measure and quantify Tyr phosphorylation in plant cells, which can also be applied to whole plants. We combined a <sup>15</sup>N stable isotope metabolic labeling strategy with an immuno-affinity purification using phospho-tyrosine (pY) specific antibodies. This single enrichment strategy was sufficient to reproducibly identify and quantify pY containing peptides from total plant cell extract in a single LC–MS/MS run. We succeeded in identifying 149 unique pY peptides originating from 135 proteins, including a large set of different protein kinases and several receptor-like kinases. We used flagellin perception by <i>Arabidopsis</i> cells, a model system for pathogen triggered immune (PTI) signaling, to test our approach. We reproducibly quantified 23 pY peptides in 2 inversely labeled biological replicates identifying 11 differentially phosphorylated proteins. These include a set of 3 well-characterized flagellin responsive MAP kinases and 4 novel MAP kinases. With this targeted approach, we elucidate a new level of complexity in flagellin-induced MAP kinase activation
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