Publications by authors named "Wei-Jun Qian"

192 Publications

Fate, cytotoxicity and cellular metabolomic impact of ingested nanoscale carbon dots using simulated digestion and a triculture small intestinal epithelial model.

NanoImpact 2021 Jul 13;23. Epub 2021 Aug 13.

Center for Nanotechnology and Nanotoxicology, Department of Environmental Health, Harvard School of Public Health, 655 Huntington Ave Boston, MA 02115, USA.

Carbon dots (CDs) are a promising material currently being explored in many industrial applications in the biomedical and agri-food areas; however, studies supporting the environmental health risk assessment of CDs are needed. This study focuses on various CD forms including iron (FeCD) and copper (CuCD) doped CDs synthesized using hydrothermal method, their fate in gastrointestinal tract, and their cytotoxicity and potential changes to cellular metabolome in a triculture small intestinal epithelial model. Physicochemical characterization revealed that 75% of Fe in FeCD and 95% of Cu in CuCD were dissolved during digestion. No significant toxic effects were observed for pristine CDs and FeCDs. However, CuCD induced significant dose-dependent toxic effects including decreases in TEER and cell viability, increases in cytotoxicity and ROS production, and alterations in important metabolites, including D-glucose, L-cysteine, uridine, citric acid and multiple fatty acids. These results support the current understanding that pristine CDs are relatively non-toxic and the cytotoxicity is dependent on the doping molecules.
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http://dx.doi.org/10.1016/j.impact.2021.100349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428805PMC
July 2021

Elamipretide (SS-31) treatment attenuates age-associated post-translational modifications of heart proteins.

Geroscience 2021 Sep 4. Epub 2021 Sep 4.

Department of Pathology, University of Washington, 1959 NE Pacific St, Seattle, WA, 98195, USA.

It has been demonstrated that elamipretide (SS-31) rescues age-related functional deficits in the heart but the full set of mechanisms behind this have yet to be determined. We investigated the hypothesis that elamipretide influences post-translational modifications to heart proteins. The S-glutathionylation and phosphorylation proteomes of mouse hearts were analyzed using shotgun proteomics to assess the effects of aging on these post-translational modifications and the ability of the mitochondria-targeted drug elamipretide to reverse age-related changes. Aging led to an increase in oxidation of protein thiols demonstrated by increased S-glutathionylation of cysteine residues on proteins from Old (24 months old at the start of the study) mouse hearts compared to Young (5-6 months old). This shift in the oxidation state of the proteome was almost completely reversed by 8 weeks of treatment with elamipretide. Many of the significant changes that occurred were in proteins involved in mitochondrial or cardiac function. We also found changes in the mouse heart phosphoproteome that were associated with age, some of which were partially restored with elamipretide treatment. Parallel reaction monitoring of a subset of phosphorylation sites revealed that the unmodified peptide reporting for Myot S231 increased with age, but not its phosphorylated form and that both phosphorylated and unphosphorylated forms of the peptide covering cMyBP-C S307 increased, but that elamipretide treatment did not affect these changes. These results suggest that changes to thiol redox state and phosphorylation status are two ways in which age may affect mouse heart function, which can be restored by treatment with elamipretide.
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http://dx.doi.org/10.1007/s11357-021-00447-6DOI Listing
September 2021

Mass spectrometry-based direct detection of multiple types of protein thiol modifications in pancreatic beta cells under endoplasmic reticulum stress.

Redox Biol 2021 Oct 17;46:102111. Epub 2021 Aug 17.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address:

Thiol-based post-translational modifications (PTMs) play a key role in redox-dependent regulation and signaling. Functional cysteine (Cys) sites serve as redox switches, regulated through multiple types of PTMs. Herein, we aim to characterize the complexity of thiol PTMs at the proteome level through the establishment of a direct detection workflow. The LC-MS/MS based workflow allows for simultaneous quantification of protein abundances and multiple types of thiol PTMs. To demonstrate its utility, the workflow was applied to mouse pancreatic β-cells (β-TC-6) treated with thapsigargin to induce endoplasmic reticulum (ER) stress. This resulted in the quantification of >9000 proteins and multiple types of thiol PTMs, including intra-peptide disulfide (S-S), S-glutathionylation (SSG), S-sulfinylation (SOH), S-sulfonylation (SOH), S-persulfidation (SSH), and S-trisulfidation (SSSH). Proteins with significant changes in abundance were observed to be involved in canonical pathways such as autophagy, unfolded protein response, protein ubiquitination pathway, and EIF2 signaling. Moreover, ~500 Cys sites were observed with one or multiple types of PTMs with SSH and S-S as the predominant types of modifications. In many cases, significant changes in the levels of different PTMs were observed on various enzymes and their active sites, while their protein abundance exhibited little change. These results provide evidence of independent translational and post-translational regulation of enzyme activity. The observed complexity of thiol modifications on the same Cys residues illustrates the challenge in the characterization and interpretation of protein thiol modifications and their functional regulation.
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http://dx.doi.org/10.1016/j.redox.2021.102111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8379693PMC
October 2021

A Sweet HS/HO Dual Release System and Specific Protein S-Persulfidation Mediated by Thioglucose/Glucose Oxidase.

J Am Chem Soc 2021 Aug 12;143(33):13325-13332. Epub 2021 Aug 12.

Department of Chemistry, Brown University, Providence, Rhode Island 02912, United States.

HS and HO are two redox regulating molecules that play important roles in many physiological and pathological processes. While each of them has distinct biosynthetic pathways and signaling mechanisms, the crosstalk between these two species is also known to cause critical biological responses such as protein S-persulfidation. So far, many chemical tools for the studies of HS and HO have been developed, such as the donors and sensors for HS and HO. However, these tools are normally targeting single species (e.g., only HS or only HO). As such, the crosstalk and synergetic effects between HS and HO have hardly been studied with those tools. In this work, we report a unique HS/HO dual donor system by employing 1-thio-β-d-glucose and glucose oxidase (GOx) as the substrates. This enzymatic system can simultaneously produce HS and HO in a slow and controllable fashion, without generating any bio-unfriendly byproducts. This system was demonstrated to cause efficient S-persulfidation on proteins. In addition, we expanded the system to thiolactose and thioglucose-disulfide; therefore, additional factors (β-galactosidase and cellular reductants) could be introduced to further control the release of HS/HO. This dual release system should be useful for future research on HS and HO.
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http://dx.doi.org/10.1021/jacs.1c06372DOI Listing
August 2021

Facile One-Pot Nanoproteomics for Label-Free Proteome Profiling of 50-1000 Mammalian Cells.

J Proteome Res 2021 Sep 5;20(9):4452-4461. Epub 2021 Aug 5.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99354, United States.

Recent advances in sample preparation enable label-free mass spectrometry (MS)-based proteome profiling of small numbers of mammalian cells. However, specific devices are often required to downscale sample processing volume from the standard 50-200 μL to sub-μL for effective nanoproteomics, which greatly impedes the implementation of current nanoproteomics methods by the proteomics research community. Herein, we report a facile one-pot nanoproteomics method termed SOPs-MS (urfactant-assisted ne-ot sample processing at the tandard volume coupled with MS) for convenient robust proteome profiling of 50-1000 mammalian cells. Building upon our recent development of SOPs-MS for label-free single-cell proteomics at a low μL volume, we have systematically evaluated its processing volume at 10-200 μL using 100 human cells. The processing volume of 50 μL that is in the range of volume for standard proteomics sample preparation has been selected for easy sample handling with a benchtop micropipette. SOPs-MS allows for reliable label-free quantification of ∼1200-2700 protein groups from 50 to 1000 MCF10A cells. When applied to small subpopulations of mouse colon crypt cells, SOPs-MS has revealed protein signatures between distinct subpopulation cells with identification of ∼1500-2500 protein groups for each subpopulation. SOPs-MS may pave the way for routine deep proteome profiling of small numbers of cells and low-input samples.
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http://dx.doi.org/10.1021/acs.jproteome.1c00403DOI Listing
September 2021

Tutorial: best practices and considerations for mass-spectrometry-based protein biomarker discovery and validation.

Nat Protoc 2021 08 9;16(8):3737-3760. Epub 2021 Jul 9.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.

Mass-spectrometry-based proteomic analysis is a powerful approach for discovering new disease biomarkers. However, certain critical steps of study design such as cohort selection, evaluation of statistical power, sample blinding and randomization, and sample/data quality control are often neglected or underappreciated during experimental design and execution. This tutorial discusses important steps for designing and implementing a liquid-chromatography-mass-spectrometry-based biomarker discovery study. We describe the rationale, considerations and possible failures in each step of such studies, including experimental design, sample collection and processing, and data collection. We also provide guidance for major steps of data processing and final statistical analysis for meaningful biological interpretations along with highlights of several successful biomarker studies. The provided guidelines from study design to implementation to data interpretation serve as a reference for improving rigor and reproducibility of biomarker development studies.
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http://dx.doi.org/10.1038/s41596-021-00566-6DOI Listing
August 2021

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation.

J Vis Exp 2021 Jun 21(172). Epub 2021 Jun 21.

Integrative Omics, Biological Sciences Division, Pacific Northwest National Laboratory;

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.
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http://dx.doi.org/10.3791/62671DOI Listing
June 2021

Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH.

ACS Omega 2021 May 6;6(19):12660-12666. Epub 2021 May 6.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United States.

Isobaric labeling via tandem mass tag (TMT) reagents enables sample multiplexing prior to LC-MS/MS, facilitating high-throughput large-scale quantitative proteomics. Consistent and efficient labeling reactions are essential to achieve robust quantification; therefore, embedded in our clinical proteomic protocol is a quality control (QC) sample that contains a small aliquot from each sample within a TMT set, referred to as "Mixing QC." This Mixing QC enables the detection of TMT labeling issues by LC-MS/MS before combining the full samples to allow for salvaging of poor TMT labeling reactions. While TMT labeling is a valuable tool, factors leading to poor reactions are not fully studied. We observed that relabeling does not necessarily rescue TMT reactions and that peptide samples sometimes remained acidic after resuspending in 50 mM HEPES buffer (pH 8.5), which coincided with low labeling efficiency (LE) and relatively low median reporter ion intensities (MRIIs). To obtain a more resilient TMT labeling procedure, we investigated LE, reporter ion missingness, the ratio of mean TMT set MRII to individual channel MRII, and the distribution of log 2 reporter ion ratios of Mixing QC samples. We discovered that sample pH is a critical factor in LE, and increasing the buffer concentration in poorly labeled samples before relabeling resulted in the successful rescue of TMT labeling reactions. Moreover, resuspending peptides in 500 mM HEPES buffer for TMT labeling resulted in consistently higher LE and lower missing data. By better controlling the sample pH for labeling and implementing multiple methods for assessing labeling quality before combining samples, we demonstrate that robust TMT labeling for large-scale quantitative studies is achievable.
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http://dx.doi.org/10.1021/acsomega.1c00776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154127PMC
May 2021

Repetitive diacetyl vapor exposure promotes ubiquitin proteasome stress and precedes bronchiolitis obliterans pathology.

Arch Toxicol 2021 07 24;95(7):2469-2483. Epub 2021 May 24.

Division of Pulmonology, Department of Pediatrics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 667, Rochester, NY, 14642, USA.

Bronchiolitis obliterans (BO) is a devastating lung disease seen commonly after lung transplant, following severe respiratory tract infection or chemical inhalation exposure. Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha-diketone known to cause BO when inhaled, however, the mechanisms of how inhalation exposure leads to BO development remains poorly understood. In the current work, we combined two clinically relevant models for studying the pathogenesis of DA-induced BO: (1) an in vivo rat model of repetitive DA vapor exposures with recovery and (2) an in vitro model of primary human airway epithelial cells exposed to pure DA vapors. Rats exposed to 5 consecutive days 200 parts-per-million DA 6 h per day had worsening survival, persistent hypoxemia, poor weight gain, and histologic evidence of BO 14 days after DA exposure cessation. At the end of exposure, increased expression of the ubiquitin stress protein ubiquitin-C accumulated within DA-exposed rat lung homogenates and localized primarily to the airway epithelium, the primary site of BO development. Lung proteasome activity increased concurrently with ubiquitin-C expression after DA exposure, supportive of significant proteasome stress. In primary human airway cultures, global proteomics identified 519 significantly modified proteins in DA-exposed samples relative to controls with common pathways of the ubiquitin proteasome system, endosomal reticulum transport, and response to unfolded protein pathways being upregulated and cell-cell adhesion and oxidation-reduction pathways being downregulated. Collectively, these two models suggest that diacetyl inhalation exposure causes abundant protein damage and subsequent ubiquitin proteasome stress prior to the development of chemical-induced BO pathology.
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http://dx.doi.org/10.1007/s00204-021-03076-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8336605PMC
July 2021

Enhanced epithelial sodium channel activity in neonatal Scnn1b mouse lung attenuates high oxygen-induced lung injury.

Am J Physiol Lung Cell Mol Physiol 2021 07 5;321(1):L29-L41. Epub 2021 May 5.

Division of Respiratory, Critical Care and Occupational Pulmonary Medicine, Department of Internal Medicine, University of Utah, Salt Lake City, Utah.

Prolonged oxygen therapy leads to oxidative stress, epithelial dysfunction, and acute lung injury in preterm infants and adults. Heterozygous Scnn1b mice, which overexpress lung epithelial sodium channels (ENaC), and their wild-type (WT) C57Bl6 littermates were utilized to study the pathogenesis of high fraction inspired oxygen ([Formula: see text])-induced lung injury. Exposure to high [Formula: see text] from birth to () was used to model oxidative stress. Chronic exposure of newborn pups to 85% O increased glutathione disulfide (GSSG) and elevated the GSH/GSSG redox potential () of bronchoalveolar lavage fluid (BALF). Longitudinal X-ray imaging and Evans blue-labeled-albumin assays showed that chronic 85% O and acute GSSG (400 µM) exposures decreased alveolar fluid clearance (AFC) in the WT lung. Morphometric analysis of WT pups insufflated with GSSG (400 µM) or amiloride (1 µM) showed a reduction in alveologenesis and increased lung injury compared with age-matched control pups. The Scnn1b mouse lung phenotype was not further aggravated by chronic 85% O exposure. These outcomes support the hypothesis that exposure to hyperoxia increases GSSG, resulting in reduced lung fluid reabsorption due to inhibition of amiloride-sensitive ENaC. Flavin adenine dinucleotide (FADH; 10 µM) was effective in recycling GSSG in vivo and promoted alveologenesis, but did not impact AFC nor attenuate fibrosis following high [Formula: see text] exposure. In conclusion, the data indicate that FADH may be pivotal for normal lung development, and show that ENaC is a key factor in promoting alveologenesis, sustaining AFC, and attenuating fibrotic lung injury caused by prolonged oxygen therapy in WT mice.
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http://dx.doi.org/10.1152/ajplung.00538.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8321857PMC
July 2021

Altered β-Cell Prohormone Processing and Secretion in Type 1 Diabetes.

Diabetes 2021 05 4;70(5):1038-1050. Epub 2021 May 4.

Center for Diabetes and Metabolic Diseases, Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN

Analysis of data from clinical cohorts, and more recently from human pancreatic tissue, indicates that reduced prohormone processing is an early and persistent finding in type 1 diabetes. In this article, we review the current state of knowledge regarding alterations in islet prohormone expression and processing in type 1 diabetes and consider the clinical impact of these findings. Lingering questions, including pathologic etiologies and consequences of altered prohormone expression and secretion in type 1 diabetes, and the natural history of circulating prohormone production in health and disease, are considered. Finally, key next steps required to move forward in this area are outlined, including longitudinal testing of relevant clinical populations, studies that probe the genetics of altered prohormone processing, the need for combined functional and histologic testing of human pancreatic tissues, continued interrogation of the intersection between prohormone processing and autoimmunity, and optimal approaches for analysis. Successful resolution of these questions may offer the potential to use altered prohormone processing as a biomarker to inform therapeutic strategies aimed at personalized intervention during the natural history of type 1 diabetes and as a pathogenic anchor for identification of potential disease-specific endotypes.
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http://dx.doi.org/10.2337/dbi20-0034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173804PMC
May 2021

A Comprehensive Urine Proteome Database Generated From Patients With Various Renal Conditions and Prostate Cancer.

Front Med (Lausanne) 2021 13;8:548212. Epub 2021 Apr 13.

Integrative Omics, Pacific Northwest National Laboratory, Biological Sciences Division, Richland, WA, United States.

Urine proteins can serve as viable biomarkers for diagnosing and monitoring various diseases. A comprehensive urine proteome database, generated from a variety of urine samples with different disease conditions, can serve as a reference resource for facilitating discovery of potential urine protein biomarkers. Herein, we present a urine proteome database generated from multiple datasets using 2D LC-MS/MS proteome profiling of urine samples from healthy individuals (HI), renal transplant patients with acute rejection (AR) and stable graft (STA), patients with non-specific proteinuria (NS), and patients with prostate cancer (PC). A total of ~28,000 unique peptides spanning ~2,200 unique proteins were identified with a false discovery rate of <0.5% at the protein level. Over one third of the annotated proteins were plasma membrane proteins and another one third were extracellular proteins according to gene ontology analysis. Ingenuity Pathway Analysis of these proteins revealed 349 potential biomarkers in the literature-curated database. Forty-three percentage of all known cluster of differentiation (CD) proteins were identified in the various human urine samples. Interestingly, following comparisons with five recently published urine proteome profiling studies, which applied similar approaches, there are still ~400 proteins which are unique to this current study. These may represent potential disease-associated proteins. Among them, several proteins such as serpin B3, renin receptor, and periostin have been reported as pathological markers for renal failure and prostate cancer, respectively. Taken together, our data should provide valuable information for future discovery and validation studies of urine protein biomarkers for various diseases.
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http://dx.doi.org/10.3389/fmed.2021.548212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076675PMC
April 2021

Proteomic Profiling of the Substantia Nigra to Identify Determinants of Lewy Body Pathology and Dopaminergic Neuronal Loss.

J Proteome Res 2021 05 26;20(5):2266-2282. Epub 2021 Apr 26.

Rush Alzheimer's Disease Center, Rush University Medical Center, Chicago, Illinois 60612, United States.

Proteinaceous aggregates containing α-synuclein protein called Lewy bodies in the substantia nigra is a hallmark of Parkinson's disease. The molecular mechanisms of Lewy body formation and associated neuronal loss remain largely unknown. To gain insights into proteins and pathways associated with Lewy body pathology, we performed quantitative profiling of the proteome. We analyzed substantia nigra tissue from 51 subjects arranged into three groups: cases with Lewy body pathology, Lewy body-negative controls with matching neuronal loss, and controls with no neuronal loss. Using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we characterized the proteome both in terms of protein abundances and peptide modifications. Statistical testing for differential abundance of the most abundant 2963 proteins, followed by pathway enrichment and Bayesian learning of the causal network structure, was performed to identify likely drivers of Lewy body formation and dopaminergic neuronal loss. The identified pathways include (1) Arp2/3 complex-mediated actin nucleation; (2) synaptic function; (3) poly(A) RNA binding; (4) basement membrane and endothelium; and (5) hydrogen peroxide metabolic process. According to the data, the endothelial/basement membrane pathway is tightly connected with both pathologies and likely to be one of the drivers of neuronal loss. The poly(A) RNA-binding proteins, including the ones relevant to other neurodegenerative disorders (e.g., TDP-43 and FUS), have a strong inverse correlation with Lewy bodies and may reflect an alternative mechanism of nigral neurodegeneration.
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http://dx.doi.org/10.1021/acs.jproteome.0c00747DOI Listing
May 2021

Enhancing Top-Down Proteomics of Brain Tissue with FAIMS.

J Proteome Res 2021 05 15;20(5):2780-2795. Epub 2021 Apr 15.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United States.

Proteomic investigations of Alzheimer's and Parkinson's disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein's "intact" state. Top-down proteomics (TDP) overcomes this limitation; however, it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, fractionation techniques are commonly used to reduce sample complexity. Here, we investigate gas-phase fractionation through high-field asymmetric waveform ion mobility spectrometry (FAIMS) within TDP. Utilizing a high complexity sample derived from Alzheimer's disease (AD) brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS with external compensation voltage (CV) stepping at -50, -40, and -30 CV could more than double the mean number of non-redundant proteoforms, genes, and proteome sequence coverage compared to without FAIMS. We also found that FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aβ) proteoforms, including the aggregation-prone Aβ variant which is strongly linked to AD. Raw data and associated files have been deposited to the ProteomeXchange Consortium via the MassIVE data repository with data set identifier PXD023607.
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http://dx.doi.org/10.1021/acs.jproteome.1c00049DOI Listing
May 2021

Stoichiometric Thiol Redox Proteomics for Quantifying Cellular Responses to Perturbations.

Antioxidants (Basel) 2021 Mar 23;10(3). Epub 2021 Mar 23.

Pacific Northwest National Laboratory, Biological Sciences Division, Richland, WA 99352, USA.

Post-translational modifications regulate the structure and function of proteins that can result in changes to the activity of different pathways. These include modifications altering the redox state of thiol groups on protein cysteine residues, which are sensitive to oxidative environments. While mass spectrometry has advanced the identification of protein thiol modifications and expanded our knowledge of redox-sensitive pathways, the quantitative aspect of this technique is critical for the field of redox proteomics. In this review, we describe how mass spectrometry-based redox proteomics has enabled researchers to accurately quantify the stoichiometry of reversible oxidative modifications on specific cysteine residues of proteins. We will describe advancements in the methodology that allow for the absolute quantitation of thiol modifications, as well as recent reports that have implemented this approach. We will also highlight the significance and application of such measurements and why they are informative for the field of redox biology.
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http://dx.doi.org/10.3390/antiox10030499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004825PMC
March 2021

Defective insulin receptor signaling in hPSCs skews pluripotency and negatively perturbs neural differentiation.

J Biol Chem 2021 Jan-Jun;296:100495. Epub 2021 Mar 3.

Section of Islet Cell and Regenerative Biology, Department of Medicine, Joslin Diabetes Center, Brigham and Women's Hospital, Harvard Medical School, and Harvard Stem Cell Institute, Boston, Massachusetts, USA. Electronic address:

Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. The mechanisms underlying insulin receptor (IR)-mediated signaling in the maintenance of human pluripotent stem cell (hPSC) identity and cell fate specification are not fully understood. Here, we used two independent shRNAs to stably knock down IRs in two hPSC lines that represent pluripotent stem cells and explored the consequences on expression of key proteins in pathways linked to proliferation and differentiation. We consistently observed lowered pAKT in contrast to increased pERK1/2 and a concordant elevation in pluripotency gene expression. ERK2 chromatin immunoprecipitation, luciferase assays, and ERK1/2 inhibitors established direct causality between ERK1/2 and OCT4 expression. Of importance, RNA sequencing analyses indicated a dysregulation of genes involved in cell differentiation and organismal development. Mass spectrometry-based proteomic analyses further confirmed a global downregulation of extracellular matrix proteins. Subsequent differentiation toward the neural lineage reflected alterations in SOX1PAX6 neuroectoderm and FOXG1 cortical neuron marker expression and protein localization. Collectively, our data underscore the role of IR-mediated signaling in maintaining pluripotency, the extracellular matrix necessary for the stem cell niche, and regulating cell fate specification including the neural lineage.
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http://dx.doi.org/10.1016/j.jbc.2021.100495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050001PMC
September 2021

Surfactant-assisted one-pot sample preparation for label-free single-cell proteomics.

Commun Biol 2021 03 1;4(1):265. Epub 2021 Mar 1.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.

Large numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-β-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for 'all-in-one' one-pot sample preparation. This 'all-in-one' method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics.
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http://dx.doi.org/10.1038/s42003-021-01797-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921383PMC
March 2021

Corrigendum: Near-Single-Cell Proteomics Profiling of the Proximal Tubular and Glomerulus of the Normal Human Kidney.

Front Med (Lausanne) 2020 12;7:625788. Epub 2021 Jan 12.

Division of MultiOrgan Transplantation, Department of Surgery, University of California, San Francisco, San Francisco, CA, United States.

[This corrects the article DOI: 10.3389/fmed.2020.00499.].
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http://dx.doi.org/10.3389/fmed.2020.625788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835930PMC
January 2021

Enhancement of polyhydroxyalkanoate production by co-feeding lignin derivatives with glycerol in Pseudomonas putida KT2440.

Biotechnol Biofuels 2021 Jan 7;14(1):11. Epub 2021 Jan 7.

Bioproducts, Sciences & Engineering Laboratory, Department of Biological Systems Engineering, Washington State University, Richland, WA, 99354, USA.

Background: Efficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA-producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy-co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440-for the first time, which simultaneously improved both cell biomass and PHA production.

Results: Co-feeding lignin derivatives (i.e. benzoate, vanillin, and vanillic acid) and glycerol to P. putida KT2440 was shown for the first time to simultaneously increase cell dry weight (CDW) by 9.4-16.1% and PHA content by 29.0-63.2%, respectively, compared with feeding glycerol alone. GC-MS results revealed that the addition of lignin derivatives to glycerol decreased the distribution of long-chain monomers (C10 and C12) by 0.4-4.4% and increased the distribution of short-chain monomers (C6 and C8) by 0.8-3.5%. The H-C HMBC, H-C HSQC, and H-H COSY NMR analysis confirmed that the PHA monomers (C6-C14) were produced when glycerol was fed to the bacteria alone or together with lignin derivatives. Moreover, investigation of the glycerol/benzoate/nitrogen ratios showed that benzoate acted as an independent factor in PHA synthesis. Furthermore, H, C and P NMR metabolite analysis and mass spectrometry-based quantitative proteomics measurements suggested that the addition of benzoate stimulated oxidative-stress responses, enhanced glycerol consumption, and altered the intracellular NAD/NADH and NADPH/NADP ratios by up-regulating the proteins involved in energy generation and storage processes, including the Entner-Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty acid β-oxidation, and PHA biosynthesis.

Conclusions: This work demonstrated an effective co-carbon feeding strategy to improve PHA content/yield and convert lignin derivatives into value-added products in P. putida KT2440. Co-feeding lignin break-down products with other carbon sources, such as glycerol, has been demonstrated as an efficient way to utilize biomass to increase PHA production in P. putida KT2440. Moreover, the involvement of aromatic degradation favours further lignin utilization, and the combination of proteomics and metabolomics with NMR sheds light on the metabolic and regulatory mechanisms for cellular redox balance and potential genetic targets for a higher biomass carbon conversion efficiency.
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http://dx.doi.org/10.1186/s13068-020-01861-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7792162PMC
January 2021

Characterization of cellular oxidative stress response by stoichiometric redox proteomics.

Am J Physiol Cell Physiol 2021 02 2;320(2):C182-C194. Epub 2020 Dec 2.

Integrative Omics, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington.

The thiol redox proteome refers to all proteins whose cysteine thiols are subjected to various redox-dependent posttranslational modifications (PTMs) including glutathionylation (SSG), nitrosylation (SNO), sulfenylation (SOH), and sulfhydration (SSH). These modifications can impact various aspects of protein function such as activity, binding, conformation, localization, and interactions with other molecules. To identify novel redox proteins in signaling and regulation, it is highly desirable to have robust redox proteomics methods that can provide global, site-specific, and stoichiometric quantification of redox PTMs. Mass spectrometry (MS)-based redox proteomics has emerged as the primary platform for broad characterization of thiol PTMs in cells and tissues. Herein, we review recent advances in MS-based redox proteomics approaches for quantitative profiling of redox PTMs at physiological or oxidative stress conditions and highlight some recent applications. Considering the relative maturity of available methods, emphasis will be on two types of modifications: ) total oxidation (i.e., all reversible thiol modifications), the level of which represents the overall redox state, and ) glutathionylation, a major form of reversible thiol oxidation. We also discuss the significance of stoichiometric measurements of thiol PTMs as well as future perspectives toward a better understanding of cellular redox regulatory networks in cells and tissues.
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http://dx.doi.org/10.1152/ajpcell.00040.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948008PMC
February 2021

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells.

J Vis Exp 2020 11 6(165). Epub 2020 Nov 6.

Biological Sciences Division, Pacific Northwest National Laboratory;

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.
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http://dx.doi.org/10.3791/61797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8349108PMC
November 2020

Receptor-Driven ERK Pulses Reconfigure MAPK Signaling and Enable Persistence of Drug-Adapted BRAF-Mutant Melanoma Cells.

Cell Syst 2020 11 27;11(5):478-494.e9. Epub 2020 Oct 27.

Laboratory of Systems Pharmacology, Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Targeted inhibition of oncogenic pathways can be highly effective in halting the rapid growth of tumors but often leads to the emergence of slowly dividing persister cells, which constitute a reservoir for the selection of drug-resistant clones. In BRAF melanomas, RAF and MEK inhibitors efficiently block oncogenic signaling, but persister cells emerge. Here, we show that persister cells escape drug-induced cell-cycle arrest via brief, sporadic ERK pulses generated by transmembrane receptors and growth factors operating in an autocrine/paracrine manner. Quantitative proteomics and computational modeling show that ERK pulsing is enabled by rewiring of mitogen-activated protein kinase (MAPK) signaling: from an oncogenic BRAF monomer-driven configuration that is drug sensitive to a receptor-driven configuration that involves Ras-GTP and RAF dimers and is highly resistant to RAF and MEK inhibitors. Altogether, this work shows that pulsatile MAPK activation by factors in the microenvironment generates a persistent population of melanoma cells that rewires MAPK signaling to sustain non-genetic drug resistance.
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http://dx.doi.org/10.1016/j.cels.2020.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009031PMC
November 2020

Protein thiol oxidation in the rat lung following e-cigarette exposure.

Redox Biol 2020 10 10;37:101758. Epub 2020 Oct 10.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, United States. Electronic address:

E-cigarette (e-cig) aerosols are complex mixtures of various chemicals including humectants (propylene glycol (PG) and vegetable glycerin (VG)), nicotine, and various flavoring additives. Emerging research is beginning to challenge the "relatively safe" perception of e-cigarettes. Recent studies suggest e-cig aerosols provoke oxidative stress; however, details of the underlying molecular mechanisms remain unclear. Here we used a redox proteomics assay of thiol total oxidation to identify signatures of site-specific protein thiol modifications in Sprague-Dawley rat lungs following in vivo e-cig aerosol exposures. Histologic evaluation of rat lungs exposed acutely to e-cig aerosols revealed mild perturbations in lung structure. Bronchoalveolar lavage (BAL) fluid analysis demonstrated no significant change in cell count or differential. Conversely, total lung glutathione decreased significantly in rats exposed to e-cig aerosol compared to air controls. Redox proteomics quantified the levels of total oxidation for 6682 cysteine sites representing 2865 proteins. Protein thiol oxidation and alterations by e-cig exposure induced perturbations of protein quality control, inflammatory responses and redox homeostasis. Perturbations of protein quality control were confirmed with semi-quantification of total lung polyubiquitination and 20S proteasome activity. Our study highlights the importance of redox control in the pulmonary response to e-cig exposure and the utility of thiol-based redox proteomics as a tool for elucidating the molecular mechanisms underlying this response.
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http://dx.doi.org/10.1016/j.redox.2020.101758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575796PMC
October 2020

Near-Single-Cell Proteomics Profiling of the Proximal Tubular and Glomerulus of the Normal Human Kidney.

Front Med (Lausanne) 2020 17;7:499. Epub 2020 Sep 17.

Division of MultiOrgan Transplantation, Department of Surgery, University of California, San Francisco, San Francisco, CA, United States.

Molecular assessments at the single cell level can accelerate biological research by providing detailed assessments of cellular organization and tissue heterogeneity in both disease and health. The human kidney has complex multi-cellular states with varying functionality, much of which can now be completely harnessed with recent technological advances in tissue proteomics at a near single-cell level. We discuss the foundational steps in the first application of this mass spectrometry (MS) based proteomics method for analysis of sub-sections of the normal human kidney, as part of the Kidney Precision Medicine Project (KPMP). Using ~30-40 laser captured micro-dissected kidney cells, we identified more than 2,500 human proteins, with specificity to the proximal tubular (PT; = 25 proteins) and glomerular (Glom; = 67 proteins) regions of the kidney and their unique metabolic functions. This pilot study provides the roadmap for application of our near-single-cell proteomics workflow for analysis of other renal micro-compartments, on a larger scale, to unravel perturbations of renal sub-cellular function in the normal kidney as well as different etiologies of acute and chronic kidney disease.
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http://dx.doi.org/10.3389/fmed.2020.00499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533534PMC
September 2020

Stochiometric quantification of the thiol redox proteome of macrophages reveals subcellular compartmentalization and susceptibility to oxidative perturbations.

Redox Biol 2020 09 21;36:101649. Epub 2020 Jul 21.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. Electronic address:

Posttranslational modifications of protein cysteine thiols play a significant role in redox regulation and the pathogenesis of human diseases. Herein, we report the characterization of the cellular redox landscape in terms of quantitative, site-specific occupancies of both S-glutathionylation (SSG) and total reversible thiol oxidation (total oxidation) in RAW 264.7 macrophage cells under basal conditions. The occupancies of thiol modifications for ~4000 cysteine sites were quantified, revealing a mean site occupancy of 4.0% for SSG and 11.9% for total oxidation, respectively. Correlations between site occupancies and structural features such as pKa, relative residue surface accessibility, and hydrophobicity were observed. Proteome-wide site occupancy analysis revealed that the average occupancies of SSG and total oxidation in specific cellular compartments correlate well with the expected redox potentials of respective organelles in macrophages, consistent with the notion of redox compartmentalization. The lowest average occupancies were observed in more reducing organelles such as the mitochondria (non-membrane) and nucleus, while the highest average occupancies were found in more oxidizing organelles such as endoplasmic reticulum (ER) and lysosome. Furthermore, a pattern of subcellular susceptibility to redox changes was observed under oxidative stress induced by exposure to engineered metal oxide nanoparticles. Peroxisome, ER, and mitochondria (membrane) are the organelles which exhibit the most significant redox changes; while mitochondria (non-membrane) and Golgi were observed as the organelles being most resistant to oxidative stress. Finally, it was observed that Cys residues at enzymatic active sites generally had a higher level of occupancy compared to non-active Cys residues within the same proteins, suggesting site occupancy as a potential indicator of protein functional sites. The raw data are available via ProteomeXchange with identifier PXD019913.
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http://dx.doi.org/10.1016/j.redox.2020.101649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397701PMC
September 2020

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice.

Elife 2020 07 10;9. Epub 2020 Jul 10.

Department of Pathology, University of Washington, Seattle, United States.

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.
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http://dx.doi.org/10.7554/eLife.55513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377906PMC
July 2020

Proteomic Tissue-Based Classifier for Early Prediction of Prostate Cancer Progression.

Cancers (Basel) 2020 May 17;12(5). Epub 2020 May 17.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.

Although ~40% of screen-detected prostate cancers (PCa) are indolent, advanced-stage PCa is a lethal disease with 5-year survival rates around 29%. Identification of biomarkers for early detection of aggressive disease is a key challenge. Starting with 52 candidate biomarkers, selected from existing PCa genomics datasets and known PCa driver genes, we used targeted mass spectrometry to quantify proteins that significantly differed in primary tumors from PCa patients treated with radical prostatectomy (RP) across three study outcomes: (i) metastasis ≥1-year post-RP, (ii) biochemical recurrence ≥1-year post-RP, and (iii) no progression after ≥10 years post-RP. Sixteen proteins that differed significantly in an initial set of 105 samples were evaluated in the entire cohort (n = 338). A five-protein classifier which combined FOLH1, KLK3, TGFB1, SPARC, and CAMKK2 with existing clinical and pathological standard of care variables demonstrated significant improvement in predicting distant metastasis, achieving an area under the receiver-operating characteristic curve of 0.92 (0.86, 0.99, = 0.001) and a negative predictive value of 92% in the training/testing analysis. This classifier has the potential to stratify patients based on risk of aggressive, metastatic PCa that will require early intervention compared to low risk patients who could be managed through active surveillance.
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http://dx.doi.org/10.3390/cancers12051268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281161PMC
May 2020

Block Design with Common Reference Samples Enables Robust Large-Scale Label-Free Quantitative Proteome Profiling.

J Proteome Res 2020 07 22;19(7):2863-2872. Epub 2020 May 22.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United States.

Label-free quantitative proteomics has become an increasingly popular tool for profiling global protein abundances. However, one major limitation is the potential performance drift of the LC-MS platform over time, which, in turn, limits its utility for analyzing large-scale sample sets. To address this, we introduce an experimental and data analysis scheme based on a block design with common references within each block for enabling large-scale label-free quantification. In this scheme, a large number of samples (e.g., >100 samples) are analyzed in smaller and more manageable blocks, minimizing instrument drift and variability within individual blocks. Each designated block also contains common reference samples (e.g., controls) for normalization across all blocks. We demonstrated the robustness of this approach by profiling the proteome response of human macrophage THP-1 cells to 11 engineered nanomaterials at two different doses. A total of 116 samples were analyzed in six blocks, yielding an average coverage of 4500 proteins per sample. Following a common reference-based correction, 2537 proteins were quantified with high reproducibility without any imputation of missing values from 116 data sets. The data revealed the consistent quantification of proteins across all six blocks, as illustrated by the highly consistent abundances of house-keeping proteins in all samples and the high levels of correlation among samples from different blocks. The data also demonstrated that label-free quantification is robust and accurate enough to quantify even very subtle abundance changes as well as large fold-changes. Our streamlined workflow is easy to implement and can be readily adapted to other large cohort studies for reproducible label-free proteome quantification.
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http://dx.doi.org/10.1021/acs.jproteome.0c00310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425200PMC
July 2020

Development of Multiplexed Immuno-N-Terminomics to Reveal the Landscape of Proteolytic Processing in Early Embryogenesis of .

Anal Chem 2020 04 30;92(7):4926-4934. Epub 2020 Mar 30.

Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea.

Protein expression levels are regulated through both translation and degradation mechanisms. Levels of degradation intermediates, that is, partially degraded proteins, cannot be distinguished from those of intact proteins by global proteomics analysis, which quantify total protein abundance levels. This study aimed to develop a tool for assessing the aspects of degradation regulation via proteolytic processing through a new multiplexed N-terminomics method involving selective isobaric labeling of protein N-termini and immunoaffinity capture of the labeled N-terminal peptides. Our method allows for not only identification of proteolytic cleavage sites, but also highly multiplexed quantification of proteolytic processing. We profiled a number of potential cleavage sites by signal peptidase and provided experimental confirmation of predicted cleavage sites of signal peptide. Furthermore, the present method uniquely represents the landscape of proteomic proteolytic processing rate during early embryogenesis in , revealing the underlying mechanism of stringent decay regulation of zygotically expressed proteins during early stages of embryogenesis.
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http://dx.doi.org/10.1021/acs.analchem.9b05035DOI Listing
April 2020

Accurate Identification of Deamidation and Citrullination from Global Shotgun Proteomics Data Using a Dual-Search Delta Score Strategy.

J Proteome Res 2020 04 23;19(4):1863-1872. Epub 2020 Mar 23.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99354, United States.

Proteins with deamidated/citrullinated amino acids play critical roles in the pathogenesis of many human diseases; however, identifying these modifications in complex biological samples has been an ongoing challenge. Herein we present a method to accurately identify these modifications from shotgun proteomics data generated by a deep proteome profiling study of human pancreatic islets obtained by laser capture microdissection. All MS/MS spectra were searched twice using MSGF+ database matching, with and without a dynamic +0.9840 Da mass shift modification on amino acids asparagine, glutamine, and arginine (NQR). Consequently, each spectrum generates two peptide-to-spectrum matches (PSMs) with MSGF+ scores, which were used for the Delta Score calculation. It was observed that all PSMs with positive Delta Score values were clustered with mass errors around 0 ppm, while PSMs with negative Delta Score values were distributed nearly equally within the defined mass error range (20 ppm) for database searching. To estimate false discovery rate (FDR) of modified peptides, a "target-mock" strategy was applied in which data sets were searched against a concatenated database containing "real-modified" (+0.9840 Da) and "mock-modified" (+1.0227 Da) peptide masses. The FDR was controlled to ∼2% using a Delta Score filter value greater than zero. Manual inspection of spectra showed that PSMs with positive Delta Score values contained deamidated/citrullinated fragments in their MS/MS spectra. Many citrullinated sites identified in this study were biochemically confirmed as autoimmunogenic epitopes of autoimmune diseases in literature. The results demonstrated that in situ deamidated/citrullinated peptides can be accurately identified from shotgun tissue proteomics data using this dual-search Delta Score strategy. Raw MS data is available at ProteomeXchange (PXD010150).
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http://dx.doi.org/10.1021/acs.jproteome.9b00766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217331PMC
April 2020
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