BackgroundHistidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C2-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.Methodology/Principal FindingsUsing His-HDX-MS, the pK a values and the half-lives (t 1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK a and t 1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK a, t 1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK a and t 1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.Conclusions/SignificanceOur results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.
Here, we describe the novel use of a volatile surfactant, perfluorooctanoic acid (PFOA), for shotgun proteomics. PFOA was found to solubilize membrane proteins as effectively as sodium dodecyl sulfate (SDS). PFOA concentrations up to 0.5% (w/v) did not significantly inhibit trypsin activity. The unique features of PFOA allowed us to develop a single-tube shotgun proteomics method that used all volatile chemicals that could easily be removed by evaporation prior to mass spectrometry analysis. The experimental procedures involved: 1) extraction of proteins in 2% PFOA; 2) reduction of cystine residues with triethyl phosphine and their S-alkylation with iodoethanol; 3) trypsin digestion of proteins in 0.5% PFOA; 4) removal of PFOA by evaporation; and 5) LC-MS/MS analysis of the resulting peptides. The general applicability of the method was demonstrated with the membrane preparation of photoreceptor outer segments. We identified 75 proteins from 1 µg of the tryptic peptides in a single, 1-hour, LC-MS/MS run. About 67% of the proteins identified were classified as membrane proteins. We also demonstrate that a proteolytic 18O labeling procedure can be incorporated after the PFOA removal step for quantitative proteomic experiments. The present method does not require sample clean-up devices such as solid-phase extractions and membrane filters, so no proteins/peptides are lost in any experimental steps. Thus, this single-tube shotgun proteomics method overcomes the major drawbacks of surfactant use in proteomic experiments.
Structural characterization of proteins and their antigen complexes is essential to the development of new biologicbased medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes, and significant correlation of reactivity and solvent accessible surface area.
Diabetes mellitus is well known to cause bladder dysfunction; however, the molecular mechanisms governing this process and the effects on individual tissue elements within the bladder are poorly understood, particularly in type 2 diabetes. A shotgun proteomics approach was applied to identify proteins differentially expressed between type 2 diabetic (TallyHo) and control (SWR/J) mice in the bladder smooth muscle and urothelium, separately. We were able to identify 1760 nonredundant proteins from the detrusor smooth muscle and 3169 nonredundant proteins from urothelium. Pathway and network analysis of significantly dysregulated proteins was conducted to investigate the molecular processes associated with diabetes. This pinpointed ERK1/2 signaling as a key regulatory node in the diabetes-induced pathophysiology for both tissue types. Diabetes mellitus is reaching epidemic proportions worldwide because of several factors, most notably obesity, which is attributed to increased sedentary lifestyles and decreased physical activity. This presents both economic and healthcare challenges; the U.S., China, and India have the highest disease prevalences and therefore are facing the greatest challenges (1). According to the Centers for Disease Control and Prevention, as of 2010, 25.8 million Americans have diabetes, and it is the seventh leading cause of death in the United States. Approximately 30% of U.S. adults 20 years or older and 50% of adults 75 years or older are afflicted with this debilitating disease (2). Based on current trends, it is expected that the number of patients diagnosed with diabetes will continue to climb at an alarming rate.Some complications of diabetes include heart disease and stroke, hypertension, blindness and eye problems, kidney disease, and nervous system disease. Recently, diabetic urological complications such as nephropathy, bladder dysfunction (3) and infection, incontinence, erectile dysfunction (4), and prostate hyperplasia have been receiving increasing attention, because they affect both type 1 and type 2 diabetic patients. Diabetic uropathy, which includes diabetic bladder dysfunction (DBD) 1 , sexual or erectile dysfunction, and urinary tract infection (5), has been found in more than 80% of patients with diabetes, a higher rate of incidence than either neuropathy or nephropathy, which affect 60 and 50% of From the ‡Center
Little is known about proteomic differences between pluripotent human peripheral blood monocytes (MN) and their terminally-differentiated pulmonary counterparts, alveolar macrophages (AM). To better characterize these cell populations, we performed a label-free shotgun proteomics assessment of matched AM and MN preparations from eight healthy volunteers. With an FDR of less than 0.45%, we identified 1754 proteins within AM and 1445 from MN. Comparison of the two proteomes revealed that 1239 of the proteins found in AM were shared with MN, whereas 206 proteins were uniquely identified in MN and 515 were unique to AM. Molecular and cellular functions, protein classes, development associations, and membership in physiological systems and canonical pathways were identified among the detected proteins. Analysis of biologic processes represented by these proteomes indicated that MN were most prominently enriched for proteins involved in cellular movement and immune cell trafficking. In contrast, AM were enriched for proteins involved in protein trafficking, molecular transport, and cellular assembly and organization. These findings provide a baseline proteomic resource for further studies aimed at better understanding of the functional differences between MN and AM in both health and disease.
Mycobacterium tuberculosis (Mtb) cell wall glycolipid mannose‐capped lipoarabinomannan (ManLAM) inhibits CD4+ T‐cell activation by inhibiting proximal T‐cell receptor (TCR) signaling when activated by anti‐CD3. To understand the impact of ManLAM on CD4+ T‐cell function when both the TCR–CD3 complex and major costimulator CD28 are engaged, we performed label‐free quantitative MS and network analysis. Mixed‐effect model analysis of peptide intensity identified 149 unique peptides representing 131 proteins that were differentially regulated by ManLAM in anti‐CD3‐ and anti‐CD28‐activated CD4+ T cells. Crosstalker, a novel network analysis tool identified dysregulated translation, TCA cycle, and RNA metabolism network modules. PCNA, Akt, mTOR, and UBC were found to be bridge node proteins connecting these modules of dysregulated proteins. Altered PCNA expression and cell cycle analysis showed arrest at the G2M phase. Western blot confirmed that ManLAM inhibited Akt and mTOR phosphorylation, and decreased expression of deubiquitinating enzymes Usp9x and Otub1. Decreased NF‐κB phosphorylation suggested interference with CD28 signaling through inhibition of the Usp9x‐Akt‐mTOR pathway. Thus, ManLAM induced global changes in the CD4+ T‐cell proteome by affecting Akt‐mTOR signaling, resulting in broad functional impairment of CD4+ T‐cell activation beyond inhibition of proximal TCR–CD3 signaling.
Amino acid-specific covalent labeling is well suited to probe protein structure and macromolecular interactions, especially for macromolecules and their complexes that are difficult to examine by alternative means, due to size, complexity, or instability. Here we present a detailed account of carbodiimide-based covalent labeling (with GEE tagging) applied to a glycosylated monoclonal antibody therapeutic, which represents an important class of biologic drugs. Characterization of such proteins and their antigen complexes is essential to development of new biologic-based medicines. In this study, the experiments were optimized to preserve the structural integrity of the protein, and experimental conditions were varied and replicated to establish the reproducibility and precision of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include D, E, and the C-terminus, against the experimental surface accessibility data in order to understand the accuracy of the approach in providing an unbiased assessment of structure. Data from the protein were also compared to reactivity measures of several model peptides to explain sequence or structure-based variations in reactivity. The results highlight several advantages of this approach. These include: the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling (indicating that the label does not significantly perturb the structure of the protein), the high reproducibility of replicate experiments (<2 % variation in modification extent), the similar reactivity of the 3 target probe residues (as suggested by analysis of model peptides), and the overall positive and significant correlation of reactivity and solvent accessible surface area (the latter values predicted by the homology modeling). Attenuation of reactivity, in otherwise solvent accessible probes, is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. The results are also compared with data from hydroxyl radical-mediated oxidative footprinting on the same protein, showing that complementary information is gained from the 2 approaches, although the number of target residues in carbodiimide/GEE labeling is fewer. Overall, this approach is an accurate and precise method for assessing protein structure of biologic drugs. IntroductionMethods to describe structures of proteins and their higher order complexes continue to evolve and various advances have increased speed, efficiency, and resolution of structure determination.1 Most proteins behave according to the functiondictated-by-structure principle, and their proper conformation is central to their role in processes such as enzyme catalysis, cell signaling, or ligand binding. [2][3][4] Protein drugs (biologics) are one of the most important and fastest growing classes of drugs in the commercial marketplace today. The function and efficacy...
To date much of the biomonitoring related to exposure to polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides is from middle to high income countries, including the U.S., Canada and Europe, but such data are lacking for the majority of low to middle income countries. Using data from 64 pregnant mothers who were enrolled in 2011, we aimed to assess the concentrations of the aforementioned toxins in umbilical cord blood serum of 67 Jamaican newborns. For 97 of the 100 PCB congeners and 16 of the 17 OC pesticides, all (100%) concentrations were below their respective limits of detection (LOD). Mean (standard deviation (SD)) lipid-adjusted concentrations in cord blood serum for congeners PCB-153, PCB-180, PCB-206 and total PCB were 14.25 (3.21), 7.16 (1.71), 7.30 (1.74) and 28.15 (6.03) ng/g-lipid, respectively. The means (SD) for the 4,4′-dichlorodiphenyldichloroethylene (DDE)-hexane fraction and total-DDE were 61.61 (70.78) and 61.60 (70.76) ng/g-lipid, respectively. Compared to the U.S. and Canada, the concentrations of these toxins were lower in cord-blood serum of Jamaican newborns. We discuss that these differences could be partly due to differences in dietary patterns in these countries. Despite limitations in our dataset, our results provide information on the investigated toxins in cord blood serum that could serve as a reference for Jamaican newborns.
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