39 research outputs found
Mapping Serum Albumin Adducts of the Food-Borne Carcinogen 2‑Amino-1-methyl-6-phenylimidazo[4,5‑<i>b</i>]pyridine by Data-Dependent Tandem Mass Spectrometry
2-Amino-1-methyl-6-phenylimidazoÂ[4,5-<i>b</i>]Âpyridine (PhIP) is a heterocyclic aromatic amine that is formed
during the cooking of meats. PhIP is a potential human carcinogen:
it undergoes metabolic activation to form electrophilic metabolites
that bind to DNA and proteins, including serum albumin (SA). The structures
of PhIP-SA adducts formed in vivo are unknown and require elucidation
before PhIP protein adducts can be implemented as biomarkers in human
studies. We previously examined the reaction of genotoxic N-oxidized
metabolites of PhIP with human SA in vitro and identified covalent
adducts formed at cysteine<sup>34</sup> (Cys<sup>34</sup>); however,
other adduction products were thought to occur. We have now identified
adducts of PhIP formed at multiple sites of SA reacted with isotopic
mixtures of electrophilic metabolites of PhIP and 2-amino-1-methyl-6-[<sup>2</sup>H<sub>5</sub>]-phenylimidazoÂ[4,5-<i>b</i>]Âpyridine
([<sup>2</sup>H<sub>5</sub>]-PhIP). The metabolites used for study
were 2-nitro-1-methyl-6-phenylimidazoÂ[4,5-<i>b</i>]Âpyridine
(NO<sub>2</sub>-PhIP), 2-hydroxyamino-1-methyl-6-phenylimidazoÂ[4,5-<i>b</i>]Âpyridine (HONH-PhIP), or <i>N</i>-acetyloxy-2-amino-1-methyl-6-phenylimidazoÂ[4,5-<i>b</i>]Âpyridine (<i>N</i>-acetoxy-PhIP). Following
proteolytic digestion, PhIP-adducted peptides were separated by ultra
performance liquid chromatography and characterized by ion trap mass
spectrometry, employing isotopic data-dependent scanning. Analysis
of the tryptic or tryptic/chymotryptic digests of SA modified with
NO<sub>2</sub>-PhIP revealed that adduction occurred at Cys<sup>34</sup>, Lys<sup>195</sup>, Lys<sup>199</sup>, Lys<sup>351</sup>, Lys<sup>541</sup>, Tyr<sup>138</sup>, Tyr<sup>150</sup>, Tyr<sup>401</sup>, and Tyr<sup>411</sup>, whereas the only site of HONH-PhIP adduction
was detected at Cys<sup>34</sup>. <i>N</i>-Acetoxy-PhIP,
a penultimate metabolite of PhIP that reacts with DNA to form covalent
adducts, did not appear to form stable adducts with SA; instead, PhIP
and 2-amino-1-methyl-6-(5-hydroxy)-phenylimidazoÂ[4,5-<i>b</i>]Âpyridine, an aqueous reaction product of the proposed nitrenium
ion of PhIP, were recovered during the proteolysis of <i>N</i>-acetoxy-PhIP-modified SA. Some of these SA adduction products of
PhIP may be implemented in molecular epidemiology studies to assess
the role of well-done cooked meat, PhIP, and the risk of cancer
Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype
In
our previous work, we showed that electrospray ionization of
intact polyclonal kappa and lambda light chains isolated from normal
serum generates two distinct, Gaussian-shaped, molecular mass distributions
representing the light-chain repertoire. During the analysis of a
large (>100) patient sample set, we noticed a low-intensity molecular
mass distribution with a mean of approximately 24 250 Da, roughly
800 Da higher than the mean of the typical kappa molecular-mass distribution
mean of 23 450 Da. We also observed distinct clones in this
region that did not appear to contain any typical post-translational
modifications that would account for such a large mass shift. To determine
the origin of the high molecular mass clones, we performed de novo
bottom-up mass spectrometry on a purified IgM monoclonal light chain
that had a calculated molecular mass of 24 275.03 Da. The entire
sequence of the monoclonal light chain was determined using multienzyme
digestion and de novo sequence-alignment software and was found to
belong to the germline allele IGKV2-30. The alignment of kappa germline
sequences revealed ten IGKV2 and one IGKV4 sequences that contained
additional amino acids in their CDR1 region, creating the high-molecular-mass
phenotype. We also performed an alignment of lambda germline sequences,
which showed additional amino acids in the CDR2 region, and the FR3
region of functional germline sequences that result in a high-molecular-mass
phenotype. The work presented here illustrates the ability of mass
spectrometry to provide information on the diversity of light-chain
molecular mass phenotypes in circulation, which reflects the germline
sequences selected by the immunoglobulin-secreting B-cell population
Label-Free Neuroproteomics of the Hippocampal-Accumbal Circuit Reveals Deficits in Neurotransmitter and Neuropeptide Signaling in Mice Lacking Ethanol-Sensitive Adenosine Transporter
The neural circuit of the dorsal
hippocampus (dHip) and nucleus
accumbens (NAc) contributes to cue-induced learning and addictive
behaviors, as demonstrated by the escalation of ethanol-seeking behaviors
observed following deletion of the adenosine equilibrative nucleoside
transporter 1 (ENT1<sup>–/–</sup>) in mice. Here we
perform quantitative LC–MS/MS neuroproteomics in the dHip and
NAc of ENT1<sup>–/–</sup> mice. Using Ingenuity Pathway
Analysis, we identified proteins associated with increased long-term
potentiation, ARP2/3-mediated actin cytoskeleton signaling and protein
expression patterns suggesting deficits in glutamate degradation,
GABAergic signaling, as well as significant changes in bioenergetics
and energy homeostasis (oxidative phosphorylation, TCA cycle, and
glycolysis). These pathways are consistent with previously reported
behavioral and biochemical phenotypes that typify mice lacking ENT1.
Moreover, we validated decreased expression of the SNARE complex protein
VAMP1 (synaptobrevin-1) in the dHip as well as decreased expression
of pro-dynorphin (PDYN), neuroendocrine convertase (PCSK1), and Leu-Enkephalin
(dynorphin-A) in the NAc. Taken together, our proteomic approach provides
novel pathways indicating that ENT1-regulated signaling is essential
for neurotransmitter release and neuropeptide processing, both of
which underlie learning and reward-seeking behaviors
Label-Free Neuroproteomics of the Hippocampal-Accumbal Circuit Reveals Deficits in Neurotransmitter and Neuropeptide Signaling in Mice Lacking Ethanol-Sensitive Adenosine Transporter
The neural circuit of the dorsal
hippocampus (dHip) and nucleus
accumbens (NAc) contributes to cue-induced learning and addictive
behaviors, as demonstrated by the escalation of ethanol-seeking behaviors
observed following deletion of the adenosine equilibrative nucleoside
transporter 1 (ENT1<sup>–/–</sup>) in mice. Here we
perform quantitative LC–MS/MS neuroproteomics in the dHip and
NAc of ENT1<sup>–/–</sup> mice. Using Ingenuity Pathway
Analysis, we identified proteins associated with increased long-term
potentiation, ARP2/3-mediated actin cytoskeleton signaling and protein
expression patterns suggesting deficits in glutamate degradation,
GABAergic signaling, as well as significant changes in bioenergetics
and energy homeostasis (oxidative phosphorylation, TCA cycle, and
glycolysis). These pathways are consistent with previously reported
behavioral and biochemical phenotypes that typify mice lacking ENT1.
Moreover, we validated decreased expression of the SNARE complex protein
VAMP1 (synaptobrevin-1) in the dHip as well as decreased expression
of pro-dynorphin (PDYN), neuroendocrine convertase (PCSK1), and Leu-Enkephalin
(dynorphin-A) in the NAc. Taken together, our proteomic approach provides
novel pathways indicating that ENT1-regulated signaling is essential
for neurotransmitter release and neuropeptide processing, both of
which underlie learning and reward-seeking behaviors
Image_3_Liver mesenchymal stem cells are superior inhibitors of NK cell functions through differences in their secretome compared to other mesenchymal stem cells.tiff
Liver-resident mesenchymal stem cells (L-MSCs) are superior inhibitors of alloreactive T cell responses compared to their counterparts from bone marrow (BM-MSCs) or adipose tissue (A-MSCs), suggesting a role in liver’s overall tolerogenic microenvironment. Whether L-MSCs also impact NK cell functions differently than other MSCs is not known. We generated and characterized L-MSCs, A-MSCs and BM-MSCs from human tissues. The mass spectrometry analysis demonstrated that L-MSC secretome is uniquely different than that of A-MSC/BM-MSC, with enriched protein sets involved in IFNγ responses and signaling. When co-cultured with primary human NK cells, L-MSCs but not other MSCs, decreased surface expression of activating receptors NKp44 and NKG2D. L-MSCs also decreased IFNγ secretion by IL-2-stimulated NK cells more effectively than other MSCs. Cytolytic function of NK cells were reduced significantly when co-cultured with L-MSCs, whereas A-MSCs or BM-MSCs did not have a major impact. Mechanistic studies showed that the L-MSC-mediated reduction in NK cell cytotoxicity is not through changes in secretion of the cytotoxic proteins Perforin, Granzyme A or B, but through increased production of HLA-C1 found in L-MSC secretome that inhibits NK cells by stimulating their inhibitory receptor KIRDL2/3. L-MSCs are more potent inhibitors of NK cell functions than A-MSC or BM-MSC. Combined with their T cell inhibitory features, these results suggest L-MSCs contribute to the tolerogenic liver microenvironment and liver-induced systemic tolerance often observed after liver transplantation.</p
Image_1_Liver mesenchymal stem cells are superior inhibitors of NK cell functions through differences in their secretome compared to other mesenchymal stem cells.tiff
Liver-resident mesenchymal stem cells (L-MSCs) are superior inhibitors of alloreactive T cell responses compared to their counterparts from bone marrow (BM-MSCs) or adipose tissue (A-MSCs), suggesting a role in liver’s overall tolerogenic microenvironment. Whether L-MSCs also impact NK cell functions differently than other MSCs is not known. We generated and characterized L-MSCs, A-MSCs and BM-MSCs from human tissues. The mass spectrometry analysis demonstrated that L-MSC secretome is uniquely different than that of A-MSC/BM-MSC, with enriched protein sets involved in IFNγ responses and signaling. When co-cultured with primary human NK cells, L-MSCs but not other MSCs, decreased surface expression of activating receptors NKp44 and NKG2D. L-MSCs also decreased IFNγ secretion by IL-2-stimulated NK cells more effectively than other MSCs. Cytolytic function of NK cells were reduced significantly when co-cultured with L-MSCs, whereas A-MSCs or BM-MSCs did not have a major impact. Mechanistic studies showed that the L-MSC-mediated reduction in NK cell cytotoxicity is not through changes in secretion of the cytotoxic proteins Perforin, Granzyme A or B, but through increased production of HLA-C1 found in L-MSC secretome that inhibits NK cells by stimulating their inhibitory receptor KIRDL2/3. L-MSCs are more potent inhibitors of NK cell functions than A-MSC or BM-MSC. Combined with their T cell inhibitory features, these results suggest L-MSCs contribute to the tolerogenic liver microenvironment and liver-induced systemic tolerance often observed after liver transplantation.</p
Image_2_Liver mesenchymal stem cells are superior inhibitors of NK cell functions through differences in their secretome compared to other mesenchymal stem cells.tiff
Liver-resident mesenchymal stem cells (L-MSCs) are superior inhibitors of alloreactive T cell responses compared to their counterparts from bone marrow (BM-MSCs) or adipose tissue (A-MSCs), suggesting a role in liver’s overall tolerogenic microenvironment. Whether L-MSCs also impact NK cell functions differently than other MSCs is not known. We generated and characterized L-MSCs, A-MSCs and BM-MSCs from human tissues. The mass spectrometry analysis demonstrated that L-MSC secretome is uniquely different than that of A-MSC/BM-MSC, with enriched protein sets involved in IFNγ responses and signaling. When co-cultured with primary human NK cells, L-MSCs but not other MSCs, decreased surface expression of activating receptors NKp44 and NKG2D. L-MSCs also decreased IFNγ secretion by IL-2-stimulated NK cells more effectively than other MSCs. Cytolytic function of NK cells were reduced significantly when co-cultured with L-MSCs, whereas A-MSCs or BM-MSCs did not have a major impact. Mechanistic studies showed that the L-MSC-mediated reduction in NK cell cytotoxicity is not through changes in secretion of the cytotoxic proteins Perforin, Granzyme A or B, but through increased production of HLA-C1 found in L-MSC secretome that inhibits NK cells by stimulating their inhibitory receptor KIRDL2/3. L-MSCs are more potent inhibitors of NK cell functions than A-MSC or BM-MSC. Combined with their T cell inhibitory features, these results suggest L-MSCs contribute to the tolerogenic liver microenvironment and liver-induced systemic tolerance often observed after liver transplantation.</p
Table_1_Liver mesenchymal stem cells are superior inhibitors of NK cell functions through differences in their secretome compared to other mesenchymal stem cells.docx
Liver-resident mesenchymal stem cells (L-MSCs) are superior inhibitors of alloreactive T cell responses compared to their counterparts from bone marrow (BM-MSCs) or adipose tissue (A-MSCs), suggesting a role in liver’s overall tolerogenic microenvironment. Whether L-MSCs also impact NK cell functions differently than other MSCs is not known. We generated and characterized L-MSCs, A-MSCs and BM-MSCs from human tissues. The mass spectrometry analysis demonstrated that L-MSC secretome is uniquely different than that of A-MSC/BM-MSC, with enriched protein sets involved in IFNγ responses and signaling. When co-cultured with primary human NK cells, L-MSCs but not other MSCs, decreased surface expression of activating receptors NKp44 and NKG2D. L-MSCs also decreased IFNγ secretion by IL-2-stimulated NK cells more effectively than other MSCs. Cytolytic function of NK cells were reduced significantly when co-cultured with L-MSCs, whereas A-MSCs or BM-MSCs did not have a major impact. Mechanistic studies showed that the L-MSC-mediated reduction in NK cell cytotoxicity is not through changes in secretion of the cytotoxic proteins Perforin, Granzyme A or B, but through increased production of HLA-C1 found in L-MSC secretome that inhibits NK cells by stimulating their inhibitory receptor KIRDL2/3. L-MSCs are more potent inhibitors of NK cell functions than A-MSC or BM-MSC. Combined with their T cell inhibitory features, these results suggest L-MSCs contribute to the tolerogenic liver microenvironment and liver-induced systemic tolerance often observed after liver transplantation.</p
Pepitome: Evaluating Improved Spectral Library Search for Identification Complementarity and Quality Assessment
Spectral libraries have emerged as a viable alternative
to protein
sequence databases for peptide identification. These libraries contain
previously detected peptide sequences and their corresponding tandem
mass spectra (MS/MS). Search engines can then identify peptides by
comparing experimental MS/MS scans to those in the library. Many of
these algorithms employ the dot product score for measuring the quality
of a spectrum-spectrum match (SSM). This scoring system does not offer
a clear statistical interpretation and ignores fragment ion <i>m</i>/<i>z</i> discrepancies in the scoring. We developed
a new spectral library search engine, Pepitome, which employs statistical
systems for scoring SSMs. Pepitome outperformed the leading library
search tool, SpectraST, when analyzing data sets acquired on three
different mass spectrometry platforms. We characterized the reliability
of spectral library searches by confirming shotgun proteomics identifications
through RNA-Seq data. Applying spectral library and database searches
on the same sample revealed their complementary nature. Pepitome identifications
enabled the automation of quality analysis and quality control (QA/QC)
for shotgun proteomics data acquisition pipelines
Integrated transcriptomic and proteomic analysis of the molecular cargo of extracellular vesicles derived from porcine adipose tissue-derived mesenchymal stem cells
<div><p>Background</p><p>Mesenchymal stromal/stem cell (MSC) transplantation is a promising therapy for tissue regeneration. Extracellular vesicles (EVs) released by MSCs act as their paracrine effectors by delivering proteins and genetic material to recipient cells. To assess how their cargo mediates biological processes that drive their therapeutic effects, we integrated miRNA, mRNA, and protein expression data of EVs from porcine adipose tissue-derived MSCs.</p><p>Methods</p><p>Simultaneous expression profiles of miRNAs, mRNAs, and proteins were obtained by high-throughput sequencing and LC-MS/MS proteomic analysis in porcine MSCs and their daughter EVs (n = 3 each). TargetScan and ComiR were used to predict miRNA target genes. Functional annotation analysis was performed using DAVID 6.7 database to rank primary gene ontology categories for the enriched mRNAs, miRNA target genes, and proteins. STRING was used to predict associations between mRNA and miRNA target genes.</p><p>Results</p><p>Differential expression analysis revealed 4 miRNAs, 255 mRNAs, and 277 proteins enriched in EVs versus MSCs (fold change >2, p<0.05). EV-enriched miRNAs target transcription factors (TFs) and EV-enriched mRNAs encode TFs, but TF proteins are not enriched in EVs. Rather, EVs are enriched for proteins that support extracellular matrix remodeling, blood coagulation, inflammation, and angiogenesis.</p><p>Conclusions</p><p>Porcine MSC-derived EVs contain a genetic cargo of miRNAs and mRNAs that collectively control TF activity in EVs and recipient cells, as well as proteins capable of modulating cellular pathways linked to tissue repair. These properties provide the fundamental basis for considering therapeutic use of EVs in tissue regeneration.</p></div