First Penicillin-Binding Protein Occupancy Patterns of ß-Lactams and ß-Lactamase Inhibitors in Klebsiella pneumoniae.

Penicillin-binding proteins (PBPs) are the high-affinity target sites of all ß-lactam antibiotics in bacteria. It is well known that each ß-lactam covalently binds to and thereby inactivates different PBPs with various affinities. Despite ß-lactams serving as the cornerstone of our therapeutic armamentarium against Klebsiella pneumoniae, PBP binding data are missing for this pathogen. We aimed to generate the first PBP binding data on 13 chemically diverse and clinically relevant ß-lactams and ß-lactamase inhibitors in K. pneumoniae PBP binding was determined using isolated membrane fractions from K. pneumoniae strains ATCC 43816 and ATCC 13883. Binding reactions were conducted using ß-lactam concentrations from 0.0075 to 256 mg/liter (or 128 mg/liter). After ß-lactam exposure, unbound PBPs were labeled by Bocillin FL. Binding affinities (50% inhibitory concentrations [IC50]) were reported as the ß-lactam concentrations that half-maximally inhibited Bocillin FL binding. PBP occupancy patterns by ß-lactams were consistent across both strains. Carbapenems bound to all PBPs, with PBP2 and PBP4 as the highest-affinity targets (IC50, <0.0075 mg/liter). Preferential PBP2 binding was observed by mecillinam (amdinocillin; IC50, <0.0075 mg/liter) and avibactam (IC50, 2 mg/liter). Aztreonam showed high affinity for PBP3 (IC50, 0.06 to 0.12 mg/liter). Ceftazidime bound PBP3 at low concentrations (IC50, 0.06 to 0.25 mg/liter) and PBP1a/b at higher concentrations (4 mg/liter), whereas cefepime bound PBPs 1 to 4 at more even concentrations (IC50, 0.015 to 2 mg/liter). These PBP binding data on a comprehensive set of 13 clinically relevant ß-lactams and ß-lactamase inhibitors in K. pneumoniae enable, for the first time, the rational design and optimization of double ß-lactam and ß-lactam-ß-lactamase inhibitor combinations.

Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells.

The major drawback of the Baculovirus/Sf9 system for recombinant adeno-associated viral (rAAV) manufacturing is that most of the Bac-derived rAAV vector serotypes, with few exceptions, demonstrate altered capsid compositions and lower biological potencies. Here, we describe a new insect cell-based production platform utilizing attenuated Kozak sequence and a leaky ribosome scanning to achieve a serotype-specific modulation of AAV capsid proteins stoichiometry. By way of example, rAAV5 and rAAV9 were produced and comprehensively characterized side by side with HEK293-derived vectors. A mass spectrometry analysis documented a 3-fold increase in both viral protein (VP)1 and VP2 capsid protein content compared with human cell-derived vectors. Furthermore, we conducted an extensive analysis of encapsidated single-stranded viral DNA using next-generation sequencing and show a 6-fold reduction in collaterally packaged contaminating DNA for rAAV5 produced in insect cells. Consequently, the re-designed rAAVs demonstrated significantly higher biological potencies, even in a comparison with HEK293-manufactured rAAVs mediating, in the case of rAAV5, 4-fold higher transduction of brain tissues in mice. Thus, the described system yields rAAV vectors of superior infectivity and higher genetic identity providing a scalable platform for good manufacturing practice (GMP)-grade vector production.

Ex Vivo Application of Secreted Metabolites Produced by Soil-Inhabiting Bacillus spp. Efficiently Controls Foliar Diseases Caused by Alternaria spp.

Bacterial biological control agents (BCAs) are largely used as live products to control plant pathogens. However, due to variable environmental and ecological factors, live BCAs usually fail to produce desirable results against foliar pathogens. In this study, we investigated the potential of cell-free culture filtrates of 12 different bacterial BCAs isolated from flower beds for controlling foliar diseases caused by Alternaria spp. In vitro studies showed that culture filtrates from two isolates belonging to Bacillus subtilis and Bacillus amyloliquefaciens displayed strong efficacy and potencies against Alternaria spp. The antimicrobial activity of the culture filtrate of these two biological control agents was effective over a wider range of pH (3.0 to 9.0) and was not affected by autoclaving or proteolysis. Comparative liquid chromatography-mass spectrometry (LC-MS) analyses showed that a complex mixture of cyclic lipopeptides, primarily of the fengycin A and fengycin B families, was significantly higher in these two BCAs than inactive Bacillus spp. Interaction studies with mixtures of culture filtrates of these two species revealed additive activity, suggesting that they produce similar products, which was confirmed by LC-tandem MS analyses. In in planta pre- and postinoculation trials, foliar application of culture filtrates of B. subtilis reduced lesion sizes and lesion frequencies caused by Alternaria alternata by 68 to 81%. Taken together, our studies suggest that instead of live bacteria, culture filtrates of B. subtilis and B. amyloliquefaciens can be applied either individually or in combination for controlling foliar diseases caused by Alternaria species.

Comparative host protein interactions with HTLV-1 p30 and HTLV-2 p28: insights into difference in pathobiology of human retroviruses.

BACKGROUND: Human T lymphotropic virus type-1 (HTLV-1) and type 2 (HTLV-2) are closely related human retroviruses, but have unique disease associations. HTLV-1 is the causative agent of an aggressive T-cell leukemia known as adult T-cell leukemia (ATL), HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases. HTLV-2 infection has not been clearly associated with any disease condition. Although both viruses can transform T cells in vitro, the HTLV-1 provirus is mainly detected in CD4+ T cells whereas HTLV-2 is mainly detected in CD8+ T cells of infected individuals. HTLV-1 and HTLV-2 encode accessory proteins p30 and p28, respectively, which share partial amino acid homology and are required for viral persistence in vivo. The goal of this study was to identify host proteins interacting with p30 and p28 in order to understand their role in pathogenesis. RESULTS: Affinity-tag purification coupled with mass spectrometric (MS) analyses revealed 42 and 22 potential interacting cellular partners of p30 and p28, respectively. Of these, only three cellular proteins, protein arginine methyltransferase 5 (PRMT5), hnRNP K and 60 S ribosomal protein L8 were detected in both p30 and p28 fractions. To validate the proteomic results, four interacting proteins were selected for further analyses using immunoblot assays. In full agreement with the MS analysis two cellular proteins REGγ and NEAF-interacting protein 30 (NIP30) selectively interacted with p30 and not with p28; heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) bound to p28 and not to p30; and PRMT5 interacted with both p30 and p28. Further studies demonstrated that reduced levels of PRMT5 resulted in decreased HTLV-2 viral gene expression whereas the viral gene expression of HTLV-1 was unchanged. CONCLUSION: The comparisons of p30 and p28 host protein interaction proteome showed striking differences with some degree of overlap. PRMT5, one of the host proteins that interacted with both p30 and p28 differentially affected HTLV-1 and HTLV-2 viral gene expression suggesting that PRMT5 is involved at different stages of HTLV-1 and HTLV-2 biology. These findings suggest that distinct host protein interaction profiles of p30 and p28 could, in part, be responsible for differences in HTLV-1 and HTLV-2 pathobiology. This study provides new avenues of investigation into mechanisms of viral infection, tropism and persistence.

Top-down mass spectrometry of intact phosphorylated ß-casein: Correlation between the precursor charge state and internal fragments.

Phosphorylated proteins play essential roles in many cellular processes, and identification and characterization of the relevant phosphoproteins can help to understand underlying mechanisms. Herein, we report a collision-induced dissociation top-down approach for characterizing phosphoproteins on a quadrupole time-of-flight mass spectrometer. ß-casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact ß-casein ions with charged states up to 36+ were detected. Tandem mass spectrometry was performed on ß-casein ions of different charge states (12+ , and 15+ to 28+ ) in order to determine the effects of charge state on dissociation of this protein. Most of the abundant fragments corresponded to y, b ions, and internal fragments caused by cleavage of the N-terminal amide bond adjacent to proline residues (Xxx-Pro). The abundance of internal fragments increased with the charge state of the protein precursor ion; these internal fragments predominantly arose from one or two Xxx-Pro cleavage events and were difficult to accurately assign. The presence of abundant sodium adducts of ß-casein further complicated the spectra. Our results suggest that when interpreting top-down mass spectra of phosphoproteins and other proteins, researchers should consider the potential formation of internal fragments and sodium adducts for reliable characterization.

Toxicology and Pharmacology of an AAV Vector Expressing Codon-Optimized RPGR in RPGR-Deficient Rd9 Mice.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated AAV2tYF-GRK1-RPGRco, to treat retinitis pigmentosa (RP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (RPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1, GRK1) and is packaged in an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a safety and potency study of this vector administered by subretinal a injection in the naturally occurring RPGR-deficient Rd9 mouse model. Sixty Rd9 mice (20 per group) received a subretinal injection in the right eye of vehicle (control) or AAV2tYF-GRK1-RPGRco at one of two dose levels (4 × 108 or 4 × 109 vg/eye) and were followed for 12 weeks after injection. Vector injections were well tolerated, with no systemic toxicity. There was a trend towards reduced electroretinography b-wave amplitudes in the high vector dose group that was not statistically significant. There were no clinically important changes in hematology or clinical chemistry parameters and no vector-related ocular changes in life or by histological examination. Dose-dependent RPGR protein expression, mainly in the inner segment of photoreceptors and the adjacent connecting cilium region, was observed in all vector-treated eyes examined. Sequence integrity of the codon-optimized RPGR was confirmed by sequencing of PCR-amplified DNA, or cDNA reverse transcribed from total RNA extracted from vector-treated retinal tissues, and by sequencing of RPGR protein obtained from transfected HEK 293 cells. These results support the use of rAAV2tYF-GRK1-RPGRco in clinical studies in patients with XLRP caused by RPGR mutations.

Expression Profiling of Nonpolar Lipids in Meibum From Patients With Dry Eye: A Pilot Study.

Purpose: The purpose of this investigation was to characterize differentially expressed lipids in meibum samples from patients with dry eye disease (DED) in order to better understand the underlying pathologic mechanisms. Methods: Meibum samples were collected from postmenopausal women with DED (PW-DED; n = 5) and a control group of postmenopausal women without DED (n = 4). Lipid profiles were analyzed by direct infusion full-scan electrospray ionization mass spectrometry (ESI-MS). An initial analysis of 145 representative peaks from four classes of lipids in PW-DED samples revealed that additional manual corrections for peak overlap and isotopes only slightly affected the statistical analysis. Therefore, analysis of uncorrected data, which can be applied to a greater number of peaks, was used to compare more than 500 lipid peaks common to PW-DED and control samples. Statistical analysis of peak intensities identified several lipid species that differed significantly between the two groups. Data from contact lens wearers with DED (CL-DED; n = 5) were also analyzed. Results: Many species of the two types of diesters (DE) and very long chain wax esters (WE) were decreased by ∼20% in PW-DED, whereas levels of triacylglycerols were increased by an average of 39% ± 3% in meibum from PW-DED compared to that in the control group. Approximately the same reduction (20%) of similar DE and WE was observed for CL-DED. Conclusions: Statistical analysis of peak intensities from direct infusion ESI-MS results identified differentially expressed lipids in meibum from dry eye patients. Further studies are warranted to support these findings.

Compositional Analysis of Wax Esters in Human Meibomian Gland Secretions by Direct Infusion Electrospray Ionization Mass Spectrometry.

Wax esters (WE) are one of the predominant lipid types in human meibomian gland secretions (meibum) and account for 40-50 % of total meibum lipids. Recently, we managed to quantify 51 isomeric groups of intact WE in normal human meibum samples by direct infusion electrospray ionization mass spectrometry (ESI-MS), with each WE peak in the MS spectrum corresponding to one isomeric group (Chen et al, Invest Ophthalmol Vis Sci 54(8):5730-53, 2013). However, the information of the isomeric composition in each group was not obtained. In this study, tandem mass spectrometry (MS/MS) was applied to quantify relative amounts of these isomers using the intensities of the corresponding diagnostic ions after appropriate correction and normalization. This data was combined with the previous obtained mole fraction of each isomeric group to total WE in human meibum to determine the corresponding percentage of each isomer. A total of 23 of the most abundant WE peaks of different molecular weights (corresponding to 85.3 % of the total amount of WE) in human meibum were studied and resulted in quantification of 92 WE species. The quantitative information of composition of WE in human meibum will help better understand their role in the tear film.

Characterization of Wax Esters by Electrospray Ionization Tandem Mass Spectrometry: Double Bond Effect and Unusual Product Ions.

A series of different types of wax esters (represented by RCOOR') were systematically studied by using electrospray ionization (ESI) collision-induced dissociation tandem mass spectrometry (MS/MS) along with pseudo MS(3) (in-source dissociation combined with MS/MS) on a quadrupole time-of-flight (Q-TOF) mass spectrometer. The tandem mass spectra patterns resulting from dissociation of ammonium/proton adducts of these wax esters were influenced by the wax ester type and the collision energy applied. The product ions [RCOOH2](+), [RCO](+) and [RCO-H2O](+) that have been reported previously were detected; however, different primary product ions were demonstrated for the three wax ester types including: (1) [RCOOH2](+) for saturated wax esters, (2) [RCOOH2](+), [RCO](+) and [RCO-H2O](+) for unsaturated wax esters containing only one double bond in the fatty acid moiety or with one additional double bond in the fatty alcohol moiety, and (3) [RCOOH2](+) and [RCO](+) for unsaturated wax esters containing a double bond in the fatty alcohol moiety alone. Other fragments included [R'](+) and several series of product ions for all types of wax esters. Interestingly, unusual product ions were detected, such as neutral molecule (including water, methanol and ammonia) adducts of [RCOOH2](+) ions for all types of wax esters and [R'-2H](+) ions for unsaturated fatty acyl-containing wax esters. The patterns of tandem mass spectra for different types of wax esters will inform future identification and quantification approaches of wax esters in biological samples as supported by a preliminary study of quantification of isomeric wax esters in human meibomian gland secretions.

Proteomic analysis of cerebrospinal fluid in canine cervical spondylomyelopathy.

STUDY DESIGN: Prospective study. OBJECTIVE: To identify proteins with differential expression in the cerebrospinal fluid (CSF) from 15 clinically normal (control) dogs and 15 dogs with cervical spondylomyelopathy (CSM). SUMMARY OF BACKGROUND DATA: Canine CSM is a spontaneous, chronic, compressive cervical myelopathy similar to human cervical spondylotic myelopathy. There is a limited knowledge of the molecular mechanisms underlying these conditions. Differentially expressed CSF proteins may contribute with novel information about the disease pathogenesis in both dogs and humans. METHODS: Protein separation was performed with 2-dimensional electrophoresis. A Student t test was used to detect significant differences between groups (P < 0.05). Three comparisons were made: (1) control versus CSM-affected dogs, (2) control versus non-corticosteroid-treated CSM-affected dogs, and (3) non-corticosteroid-treated CSM-affected versus corticosteroid-treated CSM-affected dogs. Protein spots exhibiting at least a statistically significant 1.25-fold change between groups were selected for subsequent identification with capillary-liquid chromatography tandem mass spectrometry. RESULTS: A total of 96 spots had a significant average change of at least 1.25-fold in 1 of the 3 comparisons. Compared with the CSF of control dogs, CSM-affected dogs demonstrated increased CSF expression of 8 proteins including vitamin D-binding protein, gelsolin, creatine kinase B-type, angiotensinogen, α-2-HS-glycoprotein, SPARC (secreted protein, acidic, rich in cysteine), calsyntenin-1, and complement C3, and decreased expression of pigment epithelium-derived factor, prostaglandin-H2 D-isomerase, apolipoprotein E, and clusterin. In the CSF of CSM-affected dogs, corticosteroid treatment increased the expression of haptoglobin, transthyretin isoform 2, cystatin C-like, apolipoprotein E, and clusterin, and decreased the expression of angiotensinogen, α-2-HS-glycoprotein, and gelsolin. CONCLUSION: Many of the differentially expressed proteins are associated with damaged neural tissue, bone turnover, and/or compromised blood-spinal cord barrier. The knowledge of the protein changes that occur in CSM and upon corticosteroid treatment of CSM-affected patients will aid in further understanding the pathomechanisms underlying this disease. LEVEL OF EVIDENCE: N/A.

Improving patient care via development of a protein-based diagnostic test for microbe-specific detection of chronic rhinosinusitis.

OBJECTIVES/HYPOTHESIS: The hypothesis is that signature bacterial proteins can be identified in sinus secretions via high-throughput, proteomic based techniques. Nontypeable Haemophilus influenzae (NTHI) is the most common bacterial pathogen associated with sinusitis and serves as proof of principle pathogen for identifying biomarkers. STUDY DESIGN: In vitro and in vivo studies using proteomic-based analysis of cultures of NTHI and a novel, experimental chinchilla polymicrobial sinusitis model. METHODS: Nano-liquid chromatography /tandem mass spectrometry (nano-LC-MS/MS) was performed to annotate the secretome from an NTHI biofilm. A model of NTHI-induced sinusitis was developed in a chinchilla, and NTHI proteins were detected in chinchilla secretions. A reference standard RT-PCR-based assay was adapted to allow for sensitivity and specificity testing of the identified signature biomarkers in human patients. RESULTS: Outer membrane proteins P2 (OMP-P2) and P5 (OMP-P5) were identified as promising candidates for the detection of NTHI biofilms and positively detected in nasopharyngeal secretions of chinchillas experimentally infected with NTHI. An RT-PCR based test for the presence of NTHI biofilms demonstrated 100% sensitivity and 100% specificity when tested against eight unique strains commonly found in human bacterial rhinosinusitis. CONCLUSIONS: Proteomic analysis was successful in identifying signature proteins for possible use as a biomarker for chronic rhinosinusitis (CRS). OMP-P2 and OMP-P5 were validated as promising candidates and were positively detected from nasopharyngeal secretions from chinchillas experimentally infected with NTHI. Collectively, these data support the use of OMP-P2 and OMP-P5 as biomarkers for a human clinical trial to develop a point-of-care medical diagnostic test to assist in the diagnosis and treatment of CRS.

Quantitative profiling of major neutral lipid classes in human meibum by direct infusion electrospray ionization mass spectrometry.

PURPOSE: The purpose of this investigation was to better understand lipid composition in human meibum. METHODS: Intact lipids in meibum samples were detected by direct infusion electrospray ionization mass spectrometry (ESI-MS) analysis in positive detection mode using sodium iodide (NaI) as an additive. The peak intensities of all major types of lipid species, that is, wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs) were corrected for peak overlapping and isotopic distribution; an additional ionization efficiency correction was performed for WEs and CEs, which was simplified by the observation that the corresponding ionization efficiency was primarily dependent on the specific lipid class and saturation degree of the lipids while independent of the carbon chain length. A set of WE and CE standards was spiked in meibum samples for ionization efficiency determination and absolute quantitation. RESULTS: The absolute amount (µmol/mg) for each of 51 WEs and 31 CEs in meibum samples was determined. The summed masses for 51 WEs and 31 CEs accounted for 48 ± 4% and 40 ± 2%, respectively, of the total meibum lipids. The mass percentages of saturated and unsaturated species were determined to be 75 ± 2% and 25 ± 1% for CEs and 14 ± 1% and 86 ± 1% for WEs. The profiles for two types of DEs were also obtained, which include 42 α,ω Type II DEs, and 21 ω Type I-St DEs. CONCLUSIONS: Major neutral lipid classes in meibum samples were quantitatively profiled by ESI-MS analysis with NaI additive.

Oxidative modifications of mitochondria complex II.

Increased superoxide (O2 (·-)) and nitric oxide (NO) production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In the complex II, oxidative impairment, decreased protein S-glutathionylation, and increased protein tyrosine nitration at the 70 kDa subunit occur in the post-ischemic myocardium (Zhang et al., Biochemistry 49:2529-2539, 2010; Chen et al., J Biol Chem 283:27991-28003, 2008; Chen et al., J Biol Chem 282: 32640-32654, 2007). To gain the deeper insights into ROS-mediated oxidative modifications relevant in myocardial infarction, isolated complex II is subjected to in vitro oxidative modifications with GSSG (to induce cysteine S-glutathionylation) or OONO(-) (to induce tyrosine nitration). Here, we describe the protocol to characterize the specific oxidative modifications at the 70 kDa subunit by nano-LC/MS/MS analysis. We further demonstrate the cellular oxidative modification with protein nitration/S-glutathionylation with immunofluorescence microscopy using the antibodies against 3-nitrotyrosine/glutathione and complex II 70 kDa polypeptide (AbGSC90) in myocytes under conditions of oxidative stress.

Autocrine function of aldehyde dehydrogenase 1 as a determinant of diet- and sex-specific differences in visceral adiposity.

Mechanisms for sex- and depot-specific fat formation are unclear. We investigated the role of retinoic acid (RA) production by aldehyde dehydrogenase 1 (Aldh1a1, -a2, and -a3), the major RA-producing enzymes, on sex-specific fat depot formation. Female Aldh1a1(-/-) mice, but not males, were resistant to high-fat (HF) diet-induced visceral adipose formation, whereas subcutaneous fat was reduced similarly in both groups. Sexual dimorphism in visceral fat (VF) was attributable to elevated adipose triglyceride lipase (Atgl) protein expression localized in clusters of multilocular uncoupling protein 1 (Ucp1)-positive cells in female Aldh1a1(-/-) mice compared with males. Estrogen decreased Aldh1a3 expression, limiting conversion of retinaldehyde (Rald) to RA. Rald effectively induced Atgl levels via nongenomic mechanisms, demonstrating indirect regulation by estrogen. Experiments in transgenic mice expressing an RA receptor response element (RARE-lacZ) revealed HF diet-induced RARE activation in VF of females but not males. In humans, stromal cells isolated from VF of obese subjects also expressed higher levels of Aldh1 enzymes compared with lean subjects. Our data suggest that an HF diet mediates VF formation through a sex-specific autocrine Aldh1 switch, in which Rald-mediated lipolysis in Ucp1-positive visceral adipocytes is replaced by RA-mediated lipid accumulation. Our data suggest that Aldh1 is a potential target for sex-specific antiobesity therapy.

A pseudo MS3 approach for identification of disulfide-bonded proteins: uncommon product ions and database search.

It has previously been reported that disulfide and backbone bonds of native intact proteins can be concurrently cleaved using electrospray ionization (ESI) and collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). However, the cleavages of disulfide bonds result in different cysteine modifications in product ions, making it difficult to identify the disulfide-bonded proteins via database search. To solve this identification problem, we have developed a pseudo MS(3) approach by combining nozzle-skimmer dissociation (NSD) and CID on a quadrupole time-of-flight (Q-TOF) mass spectrometer using chicken lysozyme as a model. Although many of the product ions were similar to those typically seen in MS/MS spectra of enzymatically derived peptides, additional uncommon product ions were detected including c(i-1) ions (the i(th) residue being aspartic acid, arginine, lysine and dehydroalanine) as well as those from a scrambled sequence. The formation of these uncommon types of product ions, likely caused by the lack of mobile protons, were proposed to involve bond rearrangements via a six-membered ring transition state and/or salt bridge(s). A search of 20 pseudo MS(3) spectra against the Gallus gallus (chicken) database using Batch-Tag, a program originally designed for bottom up MS/MS analysis, identified chicken lysozyme as the only hit with the expectation values less than 0.02 for 12 of the spectra. The pseudo MS(3) approach may help to identify disulfide-bonded proteins and determine the associated post-translational modifications (PTMs); the confidence in the identification may be improved by incorporating the fragmentation characteristics into currently available search programs.

iTRAQ quantitative proteomics in the analysis of tears in dry eye patients.

PURPOSE: We analyzed the change in protein expression of tear film proteins in dry eye (DE) and non-DE (NDE) patients using isobaric tag for relative and absolute quantitation (iTRAQ) technology. METHODS: We categorized 24 participants into NDE, and mild (MDE), moderate-to-severe (MSDE), and mixed (MXDE) DE on the basis of clinical DE tests. Tear samples (n = 6 subjects/group) were collected using Schirmer's strips. Proteins were extracted from strips and were quantified using the Bradford assay. Protein from each sample was pooled as internal standard (IS), and 20 µg protein from each sample and the IS were digested and labeled with different tandem mass tag (TMT) isobaric mass tag labeling reagent. The reaction was quenched and the labeled peptides were mixed. Samples were injected for liquid chromatography-mass spectrometry (LC/MS/MS) analysis on the Orbitrap mass spectrometer. Bioinformatic analyses were performed using protein information resource (PIR). RESULTS: Combined results showed a total of 386 proteins in tears as determined by the iTRAQ experiments. An average of 163 proteins was detected in each of 6 biologic replicates. Of those, 55% were detected 6 times and 90% were detected multiple times (>2). In addition to the down-regulation of commonly reported proteins, such as lipocalin-1, lysozyme, and prolactin-inducible protein across all sub groups of DE, a number of proteins were significantly differentially regulated in MSDE and other subgroups of DE. A greater number of proteins were down-regulated in MSDE versus MDE, and the specific functions involved include response to stimulus (8 vs. 6 proteins), immune system process (6 vs. 4), regulation of biologic processes (3 vs. 3), and ion transport (2 vs. 2). CONCLUSIONS: iTRAQ is one of the newest tools for quantitative mass spectrometry in tear proteome research. Differences in the protein ratios can be detected between normal and DE patients. PIR is a useful resource to interpret pathways and functions of proteins.

Protein thiyl radical mediates S-glutathionylation of complex I.

Complex I is a critical site of O(2)(•-) production and the major host of reactive protein thiols in mitochondria. In response to oxidative stress, complex I protein thiols at the 51- and 75-kDa subunits are reversibly S-glutathionylated. The mechanism of complex I S-glutathionylation is mainly obtained from insight into GSSG-mediated thiol-disulfide exchange, which would require a dramatic decline in the GSH/GSSG ratio. Intrinsic complex I S-glutathionylation can be detected in the rat heart at a relatively high GSH/GSSG ratio (J. Chen et al., J. Biol. Chem. 285:3168-3180, 2010). Thus, we hypothesized that reactive thiyl radical is more likely to mediate protein S-glutathionylation of complex I. Here we employed immuno-spin trapping and tandem mass spectrometry (LC/MS/MS) to test the hypothesis in the 75-kDa subunit from S-glutathionylated complex I. Under the conditions of O(2)(•-) production in the presence of GSH, we detected complex I S-glutathionylation at Cys-226, Cys-367, and Cys-727 of the 75-kDa subunit. Addition of a radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), significantly decreased complex I S-glutathionylation and subsequently increased the protein radical adduct of complex I-DMPO as detected by immunoblotting using an anti-DMPO antibody. LC/MS/MS analysis indicated that Cys-226, Cys-554, and Cys-727 were involved in DMPO binding, confirming that formation of the complex I thiyl radical mediates S-glutathionylation. LC/MS/MS analysis also showed that Cys-554 and Cys-727 were S-sulfonated under conditions of O(2)(•-) generation in the absence of DMPO. In myocytes (HL-1 cell line) treated with menadione to trigger mitochondrial O(2)(•-) generation, complex I protein radical and S-glutathionylation were increased. Thus mediation of complex I S-glutathionylation by the protein thiyl radical provides a unique pathway for the redox regulation of mitochondrial function.