Mark E Mccomb

• PhD
• Retired at Boston University

Nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS) benefits untargeted metabolomics by enhancing sensitivity and integrating proteomics for the same sample. Here, we present a protocol to enable nLC-MS for dual metabolomics and proteomics. We describe steps for solid-phase micro-extraction (SPME)-assisted metabolite cleaning and enric...

High-sensitivity nanoflow liquid chromatography (nLC) is seldom employed in untargeted metabolomics because current sample preparation techniques are inefficient at preventing nanocapillary column performance degradation. Here, we describe an nLC-based tandem mass spectrometry workflow that enables seamless joint analysis and integration of metabol...

Background: We hypothesize that the poor survival outcomes of end-stage kidney disease (ESKD) patients undergoing hemodialysis are associated with a low filtering efficiency and selectivity. The current gold standard criteria using single or several markers show an inability to predict or disclose the treatment effect and disease progression accura...

High sensitivity nanoflow liquid chromatography (nLC) is seldom employed in untargeted metabolomics because current sample preparation techniques are inefficient to prevent nanocapillary column performance degradation. Here, we describe an nLC-based tandem mass spectrometry workflow that enables seamless joint analysis and integration of metabolomi...

Aims S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylatio...

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear...

Osteocytes, the bone cells embedded in the mineralized matrix, control bone modeling, and remodeling through direct contact with adjacent cells and via paracrine and endocrine factors that affect cells in the bone marrow microenvironment or distant organs. Osteocytes express numerous G protein‐coupled receptors (GPCRs) and thus mice lacking the sti...

Glutaredoxin‐1 (Glrx) is a small cytosolic enzyme that removes S‐glutathionylation, glutathione adducts of protein cysteine residues, thus modulating redox signaling and gene transcription. Although Glrx up‐regulation prevented endothelial cell (EC) migration and global Glrx transgenic mice had impaired ischemic vascularization, the effects of cell...

The cellular isoform of the prion protein (PrPC) serves as precursor to the infectious isoform (PrPSc), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu2+ ions. PrPC consists of two domains: a flexible N-terminal domain and a structured C-terminal domain. Both the physiological and...

Sirtuin-1 (SirT1) catalyzes NAD⁺-dependent protein lysine deacetylation and is a critical regulator of energy and lipid metabolism, mitochondrial biogenesis, apoptosis, and senescence. Activation of SirT1 mitigates metabolic perturbations associated with diabetes and obesity. Pharmacologic molecules, cellular redox, and nutritional states can regul...

Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of functional dopaminergic neurons in the nigrostriatal pathway in the brain. While current treatments provide only symptomatic relief, gene therapy has the potential to slow or halt the degeneration of nigrostriatal dopamine neurons in PD patients. Adeno-asso...

Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin’s lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins, undergoes phosphorylation by PKC and methylation by PRMT1. Previou...

Glycomics and glycoproteomics analyses by mass spectrometry require efficient front-end separation methods to enable deep characterization of heterogeneous glycoform populations. Chromatography methods are generally limited in their ability to resolve glycoforms using mobile phases that are compatible with online liquid chromatography-mass spectrom...

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamin...

One mode of γ-globin gene silencing involves a GATA-1/FOG-1/Mi2β repressor complex that binds to the -566 GATA site relative to the Aγ-globin gene CAP site. However, the mechanism as to how this repressor complex is assembled at -566 GATA site is unknown. In this study, we demonstrate that the O-GlcNAc processing enzymes, O-GlcNAc transferase (OGT)...

O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification on serine and threonine residues of many proteins, plays crucial regulatory roles in diverse biological events. As a nutrient sensor, O-GlcNAc modification (O-GlcNAcylation) on nuclear and cytoplasmic proteins underlies the pathology of diabetic complications, including ca...

Sickle Cell Disease (SCD), caused by a point mutation in the adult β‐globin gene, leads to chronic damage to multiple organs and stroke, as well as cardiovascular abnormalities and dysfunction. However, SCD patients are phenotypically normal if they carry compensatory mutations that result in continued expression of fetal γ‐globin. Thus, a logical...

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential c...

IPA predicted multiple protein networks associated with oxidative changes caused by catalase overexpression. The 11 ‘node’ proteins are highlighted in grey. Legend to network analysis: enzyme (diamond), transmembrane receptor (vertical oval), transcriptional regulator (horizontal oval), phosphatase (triangle), transporter (trapezoid), kinase (trian...

Cardiac mitochondrial maximal and uncoupled oxygen consumption were similar in both groups. (A) Maximal (State III) and uncoupled (oligomycin 2μM) (State IV) complex I substrate-driven oxygen consumption rate; (B) Maximal (State III) and uncoupled (oligomycin 2 μM) (State IV) complex II substrate-driven oxygen consumption rate. Data represents mean...

Multiplexed quantitation of reversible cysteine oxidation in Cat Tg and WT mice. (A) TMT-switch labeling strategy. To determine the total available (free and reducible) cysteines, half of each left ventricle was completely reduced and labeled with iodoTMT; to label only reversibly oxidized cysteines, all free cysteines were blocked by IAM during ly...

Validation of TMT-tagged peptides. (A) Peptide frequency histogram of the coefficient of variation (CV) for changes in total available cysteine thiols (●), reversibly oxidized cysteine thiols (□) and their occupancy (∆). (B) Distribution of TMT-tagged peptides. A total of 2264 peptides with modifications were detected by LC-MS/MS analysis, of which...

Complete list of proteins with a change in thiol oxidation in Cat Tg vs. WT. Accession number, gene ID, sites of modification and peptide sequences were retrieved from the Uniprot knowledgebase. Fold changes in Cat Tg vs. WT, were calculated from ratio of reporter ions for changes in total available cysteine as (m/z 129)/(m/z 127), reversibly oxidi...

Proteins with a change in thiol occupancy by >2-fold in Cat Tg vs. WT mice. Accession number, gene ID, sites of modification and peptide sequences were retrieved from the Uniprot knowledgebase. Fold changes in Cat Tg vs. WT, were calculated from ratio of reporter ions for changes in total available cysteine as (m/z 129)/(m/z 127), reversibly oxidiz...

Proteins involved in five canonical pathways predicted in IPA, with a change in thiol occupancy by >1.3-fold in Cat Tg vs. WT. Accession number, gene ID, sites of modification and peptide sequences were retrieved from the Uniprot knowledgebase. Fold changes in Cat Tg vs. WT, were calculated from ratio of reporter ions for changes in total available...

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic ag...

Hemoglobinopathies are the most common inherited disorders in humans and are thus the target of screening programs worldwide. Over the past decade, mass spectrometry (MS) has gained a more important role as a clinical means to diagnose variants, and a number of approaches have been proposed for characterization. Here we investigate the use of matri...

Regulated mitotic progression coupled with high‐fidelity chromosome segregation is crucial for normal cellular division. Phosphorylation. Although, phosphorylation plays a critical role regulating mitosis, it alone cannot account for all the complexities of mitosis. We have demonstrated that O‐GlcNAcylation is critical for proper mitotic regulation...

The Western style diet (high fat and high sucrose) has lead to an epidemic of unfavorable metabolic disorders associated with obesity, diabetes and hyperlipidemia; leading to cardiovascular disease (CVD). Our hypothesis is that poor diet causes unfavorable metabolic conditions due to changes in post‐translational modifications (PTMs) of proteins in...

Pulmonary arterial hypertension (PAH) is a disease characterized by increased pulmonary vascular resistance and remodeling. Increase in the population of vascular smooth muscle cells is among the key events contributing to the remodeling. Endothelin-1 (ET-1), a potent vasoconstrictor, is linked to the etiology and progression of PAH. Here we analyz...

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in...

O‐GlcNAc is a post‐translational modification (PTM) consisting of a single N‐acetylglucosamine moiety attached to serine and threonine residues in nuclear and cytoplasmic proteins. O‐GlcNAc levels can cycle dynamically in response to hormones, nutrients, or stress in order to regulate a multitude of cellular processes. For instance O‐GlcNAc cycling...

Using a novel cysteine thiol labeling strategy coupled with mass spectrometric analysis, we identified and quantified the changes in global reversible cysteine oxidation of proteins in the left ventricle of hearts from mice with metabolic syndrome-associated diastolic dysfunction. This phenotype was induced by feeding a high-fat, high-sucrose, type...

The identification of protein post‐translational modifications (PTMs) is an increasingly important component of proteomics and biomarker discovery, but very few tools exist for performing fast and easy characterization of global PTM changes and differential comparison of PTMs across groups of data obtained from liquid chromatography–tandem mass spe...

Pulmonary hypertension (PH), an independent risk factor for mortality in sickle cell disease (SCD), has epidemiologic links to systemic complications such as leg ulcers, priapism and renal disease suggestive of a diffuse vasculopathy. The pathogenesis of SCD related vasculopathy is unclear but altered redox biology is thought to be an important dis...

Phenotype/genotype correlation can provide important insight into the pathophysiology of hemoglobin disorders and molecular mechanisms regulating globin gene expression. We present 2 unusual patients with β-globin gene cluster deletions of 44 and 589 kb. In the first case, the findings suggest that competition for LCR interaction among β-like globi...

Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleo...

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. Accurate comparison of glycoforms and quantification of glycosites is an essential step in this direction. Mass spec...

The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model sy...

During mitosis, proteins undergo a variety of post-translational modifications (PTM) in order to alter function and localization. Our objective is to determine the interplay between O-GlcNAcylation and phosphorylation on proteins during mitotic progression. O-GlcNAc is the attachment of a single N-acetylglucosamine moiety to serine and threonine re...

Unfavorable metabolic conditions (metabolic disorders) associated with obesity, diabetes, and hyperlipidemia are major causes for cardiovascular disease (CVD). One major environmental cause of this may be attributed to poor diet, aka the American diet. The early detection and monitoring of the adverse effects of metabolic disease on the heart and v...

Global changes in reversible cysteine oxidation of cardiac proteins from mice fed a high fat, high sucrose (HFHS) diet for 8 months were quantified using novel 6-plex Iodo-based isobaric labels in a switch assay and high resolution mass spectrometry. 400 reversibly oxidized proteins were identified, 56 with cysteines exhibiting more than 1.5-fold c...

Unfavorable metabolic conditions (metabolic disorders) associated with obesity and diabetes are major causes for cardiovascular disease. Early detection of the adverse effects of metabolic disease remain elusive. We believe that nonspecific changes which occur in plasma proteins, indicators of inflammation and oxidants, may act as evidence of syste...

Global changes in reversible cysteine oxidation of cardiac proteins from mice fed a high fat, high sucrose (HFHS) diet for 8 months were quantified using novel 6‐plex Iodo‐based isobaric labels in a switch assay and high resolution mass spectrometry. 400 reversibly oxidized proteins were identified, 56 with cysteines exhibiting more than 1.5‐fold c...

During mitosis, proteins undergo a variety of post‐translational modifications (PTM) in order to alter function and localization. Our objective is to determine the interplay between O‐GlcNAcylation and phosphorylation on proteins during mitotic progression. O‐GlcNAc is the attachment of a single N‐acetylglucosamine moiety to serine and threonine re...

The C57BL/6J mouse fed a Western diet high in fat and sucrose (HFHS) develops obesity, metabolic syndrome, and cardiac dysfunction. Cardiac overexpression of catalase (CatTG), an enzyme that detoxifies cellular hydrogen peroxide, may protect heart from metabolic disease. Hydrogen peroxide introduces oxidative post‐translational modifications on cys...

Unfavorable metabolic conditions (metabolic disorders) associated with obesity, diabetes, and hyperlipidemia are major causes for cardiovascular disease (CVD). One major environmental cause of this may be attributed to poor diet, aka the American diet. The early detection and monitoring of the adverse effects of metabolic disease on the heart and v...

Unfavorable metabolic conditions (metabolic disorders) associated with obesity and diabetes are major causes for cardiovascular disease. Early detection of the adverse effects of metabolic disease remain elusive. We believe that nonspecific changes which occur in plasma proteins, indicators of inflammation and oxidants, may act as evidence of syste...

There is an increasing interest in the identification and characterization of protein posttranslational modifications (PTMs). The vast amount of information obtained within a typical differential proteomics makes the measure of PTMs challenging. We have begun to explore counting methods for differential analysis of PTM changes in proteomes. We have...

Post-translational modifications are essential in regulating the function, stability, and localization of proteins. One such modification is the O-GlcNAc modification. O-GlcNAc is the attachment of a single N-acetylglucosamine moiety to serine and threonine residues on nuclear and cytoplasmic proteins. O-GlcNAc is dynamically cycling on Ser/Thr res...

Unfavorable metabolic conditions (metabolic disorders) associated with obesity and diabetes are major causes for cardiovascular disease. One major cause may be attributed to poor diet, aka the American diet model. Early detection of the adverse effects of metabolic disease remain elusive. We believe that nonspecific changes which occur in plasma pr...

Significance: Oxidative post-translational modifications (OPTMs) have been demonstrated as contributing to cardiovascular physiology and pathophysiology. These modifications have been identified using antibodies as well as advanced proteomic methods, and the functional importance of each is beginning to be understood using transgenic and gene dele...

Post‐translational modifications (PTMs) are essential in regulating the function, stability, and localization of proteins. One such modification is the O‐GlcNAc. O‐GlcNAc is the attachment of a single N‐acetylglucosamine moiety to serine and threonine residues on nuclear and cytoplasmic proteins. The modification is dynamically processed on protein...

Post-translational modifications (PTMs) are essential in regulating the function, stability, and localization of proteins. One such modification is the O-GlcNAc. O-GlcNAc is the attachment of a single N-acetylglucosamine moiety to serine and threonine residues on nuclear and cytoplasmic proteins. The modification is dynamically processed on protein...

The biological functions of glycoconjugate glycans arise in the context of structural heterogeneity resulting from non-template driven biosynthetic reactions. Such heterogeneity is particularly apparent for the glycosaminoglycan (GAG) classes, of which heparan sulfate (HS) is of particular interest for its properties in binding to many classes of g...

Here we demonstrate a new paradigm in redox signaling, whereby oxidants resulting from metabolic stress directly alter protein palmitoylation by oxidizing reactive cysteine thiolates. In mice fed a high-fat, high-sucrose diet and in cultured endothelial cells (ECs) treated with high palmitate and high glucose (HPHG), there was decreased HRas palmit...

Type II citrate synthases (CSs), found in gram-negative bacteria such as Escherichia coli, are subject to strong and specific allosteric inhibition by NADH. Crystallographic studies have shown that the NADH binding sites are located at macromolecular contacts, so that tight binding requires the intact hexameric structure. Although other adenylates,...

Transthyretin (TTR) amyloidosis and hemoglobinopathies are the archetypes of molecular diseases where point mutation characterization is diagnostically critical. We have developed a Top-down analytical platform for variant and/or modified protein sequencing and are examining the feasibility of using this platform for the analysis of hemoglobin/TTR...

Non-porous polyurethane (PU) membranes and porous PU thin films are used as sample supports for MALDI-TOFMS. Mass spectra obtained are compared with those acquired using metal targets and the crushed matrix method. The compounds characterized are wheat proteins which consist of moderately water-soluble gliadins, and of water-insoluble low molecular...

The enzyme citrate synthase from E. coli is a protein with a molecular weight (Mr) of 47 885 Da (wild type). This enzyme has been studied extensively, and its amino acid sequence has been characterized. This model protein has been used in this work for development and validation of methods involving capillary electrophoresis (CE) and electrospray i...

RP-15

Sirtuin-1 (SIRT1) is an NAD(+)-dependent protein deacetylase that is sensitive to oxidative signals. Our purpose was to determine whether SIRT1 activity is sensitive to the low molecular weight nitrosothiol, S-nitrosoglutathione (GSNO), which can transduce oxidative signals into physiological responses. SIRT1 formed mixed disulfides with GSNO-Sepha...

The selective capture of target peptides poses a great challenge to modern chemists and biologists, especially when enriching them from proteome samples possessing extremes in concentration dynamic range and sequence diversity. While approaches based on traditional techniques such as biotin-avidin pairing offer versatile tools to design strategies...

In order that biological meaning may be derived and testable hypotheses may be built from proteomics experiments, assignments of proteins identified by mass spectrometry or other techniques must be supplemented with additional notation, such as information on known protein functions, protein-protein interactions, or biological pathway associations....

The extreme pathological diversity of non-Hodgkin's lymphomas has made their accurate histological assessment difficult. New diagnostics and treatment modalities are urgently needed for these lymphomas, particularly in drug development for cancer-specific targets. Previously, we showed that a subset of B cell lymphoma, diffuse large B cell lymphoma...

In addition to haemostasis, platelets mediate inflammation and clearance of bacteria from the bloodstream. As with platelet-platelet interactions, platelet-bacteria interactions involve cytoskeletal rearrangements and release of granular content. Stimulation of the immune Toll-like receptor 2 (TLR2) on the platelet surface, activates phosphoinositi...

Nitrite has recently emerged as an important bioactive molecule, capable of conferring cardioprotection and a variety of other benefits in the cardiovascular system and elsewhere. The mechanisms by which it accomplishes these functions remain largely unclear. To characterize the dose response and corresponding cardiac sequelae of transient systemic...

Background: Pulmonary hypertension (PH) in sickle cell anemia (SCA) is characterized by decreased nitric oxide bioavailability; which may, in part, be related to increased oxidative stress. It is possible that via protein post-translational modifications, oxidants are able to affect both protein structure and function. We hypothesized that, in pati...

In systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser...

Proteomic technologies, such as yeast twohybrid, mass spectrometry (MS), protein/ peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, i...

Proteomic technologies, such as yeast twohybrid, mass spectrometry (MS), protein/ peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, i...

Pulmonary hypertension (PH) in sickle cell anemia (SCA) is characterized by decreased nitric oxide bioavailability that might, in part, be related to oxidative stress. Oxidative post-translational modifications of plasma proteins may serve as hallmarks of disease severity and could result in altered protein function and structure. We hypothesized t...

Periodontitis is an inflammatory disease initiated by host-parasite interactions which contributes to connective tissue destruction and alveolar bone resorption. Porphyromonas gingivalis (P.g.), a black-pigmented Gram-negative anaerobic bacterium, is a major pathogen in the development and progression of periodontitis. To characterize the role that...

p21ras GTPase is the protein product of the most commonly mutated human oncogene and has been identified as a target for reactive oxygen and nitrogen species. Posttranslational modification of reactive thiols, by reversible S-glutathiolation and S-nitrosation, and potentially also by irreversible oxidation, may have significant effects on p21ras ac...

Multidimensional protein chromatography offers an alternative to gel-based separations for large-scale proteomic analyses of highly complex mixtures. However, these liquid separations divide the original mixtures into multitudes of discrete samples, each of which may require numerous steps of sample manipulation, such as fraction collection, buffer...

Large-scale mass spectrometry (MS)-based proteomic analyses require high-throughput sample preparation techniques due to the increasing numbers of samples that make up a typical proteomics experiment. Moreover, extensive sample pre-treatment steps are necessary prior to MS acquisition for even the most rapid and robust MS-based proteomics methodolo...

Pulmonary hypertension (PH) in sickle cell anemia (SCA) is characterized by decreased nitric oxide bioavailability; which may, in part, be related to increased oxidative stress. It is possible that via protein post-translational modifications, oxidants are able to affect both protein structure and function. We hypothesized that, in patients with SC...

See related article, pages 706–714 Cardiac ischemic preconditioning was first described in 1986 by Murry et al,1 who reported that brief periods of ischemia dramatically reduced myocardial infarct size caused by a subsequent, more prolonged period of ischemia. Since the initial report, the phenomenon has been shown to occur in many cell types, in...

Because of their complexity, the separation of intact proteins from complex mixtures is an important step to comparative proteomics and the identification and characterization of the proteins by mass spectrometry (MS). In the study reported, we evaluated the use of nonporous-reversed-phase (np-RP)-HPLC for intact protein separation prior to MS anal...

Introduction Oxidant-Sensitive Cys Challenges in Redox Proteomics Iodoacetamide-Based Redox Proteomics ICAT Approach to Redox Proteomics Validation of the ICAT Approach Using the Recombinant Protein Creatine Kinase ICAT Approach to the Complex Protein Mixtures Perspectives

We use affinity purification of the double bromodomain protein Brd2 to isolate a multicomponent nuclear complex from cultured cells, and apply mass spectrometry/proteomics methods to identify the participants. We then confirm by immunoblot several transcription co-activators and co-repressors, proteins of the Swi/Snf chromatin remodeling complex, w...

An approach is described for the simultaneous identification and quantitation of oxidant-sensitive cysteine thiols in a complex protein mixture using a thiol-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, USA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by th...

A broad range of mass spectrometers are used in mass spectrometry (MS)-based proteomics research. Each type of instrument possesses a unique design, data system and performance specifications, resulting in strengths and weaknesses for different types of experiments. Unfortunately, the native binary data formats produced by each type of mass spectro...