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1.  LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism 
Journal of Proteome Research  2014;13(4):1938-1956.
The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity.
doi:10.1021/pr401081b
PMCID: PMC3993954  PMID: 24555535
Caenorhabditis elegans; gene expression; mass spectrometry; metabolism; physiology; aging
2.  Enhanced FASP (eFASP) to Increase Proteome Coverage and Sample Recovery for Quantitative Proteomic Experiments 
Journal of Proteome Research  2014;13(4):1885-1895.
The integrity of quantitative proteomic experiments depends on the reliability and the robustness of the protein extraction, solubilization, and digestion methods utilized. Combinations of detergents, chaotropes, and mechanical disruption can yield successful protein preparations; however, the methods subsequently required to eliminate these added contaminants, in addition to the salts, nucleic acids, and lipids already in the sample, can result in significant sample losses and incomplete contaminant removal. A recently introduced method for proteomic sample preparation, filter-aided sample preparation (FASP), cleverly circumvents many of the challenges associated with traditional protein purification methods but is associated with significant sample loss. Presented here is an enhanced FASP (eFASP) approach that incorporates alternative reagents to those of traditional FASP, improving sensitivity, recovery, and proteomic coverage for processed samples. The substitution of 0.2% deoxycholic acid for urea during eFASP digestion increases tryptic digestion efficiency for both cytosolic and membrane proteins yet obviates needed cleanup steps associated with use of the deoxycholate sodium salt. For classic FASP, prepassivating Microcon filter surfaces with 5% TWEEN-20 reduces peptide loss by 300%. An express eFASP method uses tris(2-carboxyethyl)phosphine and 4-vinylpyridine to alkylate proteins prior to deposition on the Microcon filter, increasing alkylation specificity and speeding processing.
doi:10.1021/pr4010019
PMCID: PMC3993969  PMID: 24552128
filter-aided sample preparation; quantitative proteomics; detergent; ammonium deoxycholate; MSE
3.  Top-Down Proteomics of a Drop of Blood for Diabetes Monitoring 
Journal of Proteome Research  2014;13(3):1560-1569.
The most common markers for monitoring patients with diabetes are glucose and HbA1c, but additional markers such as glycated human serum albumin (HSA) have been identified that could address the glycation gap and bridge the time scales of glycemia between transient and 2–3 months. However, there is currently no technical platform that could measure these markers concurrently in a cost-effective manner. We have developed a new assay that is able to measure glucose, HbA1c, glycated HSA, and glycated apolipoprotein A-I (apoA-I) for monitoring of individual blood glycemia, as well as cysteinylated HSA, S-nitrosylated HbA, and methionine-oxidized apoA-I for gauging oxidative stress and cardiovascular risks, all in 5 μL of blood. The assay utilizes our proprietary multinozzle emitter array chip technology to enable the analysis of small volumes of blood, without complex sample preparation prior to the online and on-chip liquid chromatography–nanoelectrospray ionization mass spectrometry. Importantly, the assay employs top-down proteomics for more accurate quantitation of protein levels and for identification of post-translational modifications. Further, the assay provides multimarker, multitime-scale, and multicompartment monitoring of blood glycemia. Our assay readily segregates healthy controls from Type 2 diabetes patients and may have the potential to enable better long-term monitoring and disease management of diabetes.
doi:10.1021/pr401074t
PMCID: PMC3993886  PMID: 24533899
MEA; microfluidic chip; top-down proteomics; LC−MS; blood; diabetes; multimarker
4.  Improved Intensity-Based Label-Free Quantification via Proximity-Based Intensity Normalization (PIN) 
Journal of Proteome Research  2014;13(3):1281-1292.
Researchers are increasingly turning to label-free MS1 intensity-based quantification strategies within HPLC–ESI–MS/MS workflows to reveal biological variation at the molecule level. Unfortunately, HPLC–ESI–MS/MS workflows using these strategies produce results with poor repeatability and reproducibility, primarily due to systematic bias and complex variability. While current global normalization strategies can mitigate systematic bias, they fail when faced with complex variability stemming from transient stochastic events during HPLC–ESI–MS/MS analysis. To address these problems, we developed a novel local normalization method, proximity-based intensity normalization (PIN), based on the analysis of compositional data. We evaluated PIN against common normalization strategies. PIN outperforms them in dramatically reducing variance and in identifying 20% more proteins with statistically significant abundance differences that other strategies missed. Our results show the PIN enables the discovery of statistically significant biological variation that otherwise is falsely reported or missed.
doi:10.1021/pr400866r
PMCID: PMC3993879  PMID: 24571364
normalization; label-free quantification; proteomics; peptidomics; bioinformatics
5.  Assessment of MS/MS Search Algorithms with Parent-Protein Profiling 
Journal of Proteome Research  2014;13(4):1823-1832.
Peptide mass spectrometry relies crucially on algorithms that match peptides to spectra. We describe a method to evaluate the accuracy of these algorithms based on the masses of parent proteins before trypsin endoprotease digestion. Measurement of conformance to parent proteins provides a score for comparison of the performances of different algorithms as well as alternative parameter settings for a given algorithm. Tracking of conformance scores for spectrum matches to proteins with progressively lower expression levels revealed that conformance scores are not uniform within data sets but are significantly lower for less abundant proteins. Similarly peptides with lower algorithm peptide-spectrum match scores have lower conformance. Although peptide mass spectrometry data is typically filtered through decoy analysis to ensure a low false discovery rate, this analysis confirms that the filtered data should not be considered as having a uniform confidence. The analysis suggests that use of different algorithms and multiple standardized parameter settings of these algorithms can increase significantly the numbers of peptides identified. This data set can be used as a resource for future algorithm assessment.
doi:10.1021/pr401090d
PMCID: PMC3993968  PMID: 24533481
peptide mass spectrometry; trypsin; OMSSA; SEQUEST; Mascot; algorithm parameter sets; parent-protein conformance; decoy analysis
6.  Cd2+-Induced Alteration of the Global Proteome of Human Skin Fibroblast Cells 
Journal of Proteome Research  2014;13(3):1677-1687.
Cadmium (Cd2+) is a toxic heavy metal and a well-known human carcinogen. The toxic effects of Cd2+ on biological systems are diverse and thought to be exerted through a complex array of mechanisms. Despite the large number of studies aimed to elucidate the toxic mechanisms of action of Cd2+, few have been targeted toward investigating the ability of Cd2+ to disrupt multiple cellular pathways simultaneously and the overall cellular responses toward Cd2+ exposure. In this study, we employed a quantitative proteomic method, relying on stable isotope labeling by amino acids in cell culture (SILAC) and LC–MS/MS, to assess the Cd2+-induced simultaneous alterations of multiple cellular pathways in cultured human skin fibroblast cells. By using this approach, we were able to quantify 2931 proteins, and 400 of them displayed significantly changed expression following Cd2+ exposure. Our results unveiled that Cd2+ treatment led to the marked upregulation of several antioxidant enzymes (e.g., metallothionein-1G, superoxide dismutase, pyridoxal kinase, etc.), enzymes associated with glutathione biosynthesis and homeostasis (e.g., glutathione S-transferases, glutathione synthetase, glutathione peroxidase, etc.), and proteins involved in cellular energy metabolism (e.g., glycolysis, pentose phosphate pathway, and the citric acid cycle). Additionally, we found that Cd2+ treatment resulted in the elevated expression of two isoforms of dimethylarginine dimethylaminohydrolase (DDAH I and II), enzymes known to play a key role in regulating nitric oxide biosynthesis. Consistent with these findings, we observed elevated formation of nitric oxide in human skin (GM00637) and lung (IMR-90) fibroblast cells following Cd2+ exposure. The upregulation of DDAH I and II suggests a role of nitric oxide synthesis in Cd2+-induced toxicity in human cells.
doi:10.1021/pr401159f
PMCID: PMC3993958  PMID: 24527689
Cd2+; mass spectrometry; protein quantitation; stable isotope labeling by amino acids in cell culture; reactive oxygen species; nitric oxide synthesis
7.  Metabolomic analysis of the effects of chronic arsenic exposure in a mouse model of diet-induced fatty liver disease 
Journal of proteome research  2013;13(2):547-554.
Arsenic is a widely-distributed environmental component that is associated with a variety of cancer and non-cancer adverse health effects. Additional lifestyle factors, such as diet, contribute to the manifestation of disease. Recently, arsenic was found to increase inflammation and liver injury in a dietary model of fatty liver disease. The purpose of the present study was to investigate potential mechanisms of this diet-environment interaction via a high throughput metabolomics approach. GC×GC-TOF MS was used to identify metabolites that were significantly increased or decreased in the livers of mice fed a Western diet (a diet high in fat and cholesterol) and co-exposed to arsenic-contaminated drinking water. The results showed that there are distinct hepatic metabolomic profiles associated with eating a high fat diet, drinking arsenic-contaminated water, and the combination of the two. Among the metabolites that were decreased when arsenic exposure was combined with a high fat diet were short-chain and medium-chain fatty acid metabolites and the anti-inflammatory amino acid, glycine. These results are consistent with the observed increase in inflammation and cell death in the livers of these mice, and they point to potentially novel mechanisms by which these metabolic pathways could be altered by arsenic in the context of diet-induced fatty liver disease.
doi:10.1021/pr400719u
PMCID: PMC3946282  PMID: 24328084
GC×GC-TOF MS; metabolomics; liver; sodium arsenite; high fat diet
8.  Accurate Label-Free Protein Quantitation with High- and Low-Resolution Mass Spectrometers 
Journal of proteome research  2013;13(2):1034-1044.
Label-free quantitation of proteins analyzed by tandem mass spectrometry uses either integrated peak intensity from the parent-ion mass analysis (MS1) or features from fragment-ion analysis (MS2), such as spectral counts or summed fragment-ion intensity. We directly compared MS1 and MS2 quantitation by analyzing human protein standards diluted into Escherichia coli extracts on an Orbitrap mass spectrometer. We found that summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity. We compared these results to those obtained from two low-resolution ion-trap mass spectrometers; summed MS2 intensities from LTQ and LTQ Velos instruments were similar in accuracy to those from the Orbitrap. Data from all three instruments are available via ProteomeXchange with identifier PXD000602. Abundance measurements using MS1 or MS2 intensities had limitations, however. While measured protein concentration was on average well correlated with the known concentration, there was considerable protein-to-protein variation. Moreover, not all human proteins diluted to a mole fraction of 10−3 or lower were detected, with a strong fall-off below 10−4 mole fraction. These results show that MS1 and MS2 intensities are simple measures of protein abundance that are on average accurate, but should be limited to quantitation of proteins of intermediate to higher fractional abundance.
doi:10.1021/pr401017h
PMCID: PMC3946283  PMID: 24295401
Intensity; spectral counts; Orbitrap; ion trap
9.  Exosomes from myeloid derived suppressor cells carry biologically active proteins 
Journal of proteome research  2013;13(2):836-843.
Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they inhibit natural anti-tumor immunity and are an obstacle to anti-cancer immunotherapies. They mediate immune suppression through their production of proteins and soluble mediators that prevent the activation of tumor-reactive T lymphyocytes, polarize macrophages towards a tumor-promoting phenotype, and facilitate angiogenesis. The accumulation and suppressive potency of MDSC is regulated by inflammation within the tumor microenvironment. Recently exosomes have been proposed to act as intercellular communicators, carrying active proteins and other molecules between sender cells and receiver cells. In this report we describe the proteome of exosomes shed by MDSC induced in BALB/c mice by the 4T1 mammary carcinoma. Using bottom-up proteomics, we have identified 412 proteins. Spectral counting identified 63 proteins whose abundance was altered > 2-fold in the inflammatory environment. The pro-inflammatory proteins S100A8 and S100A9, previously shown to be secreted by MDSC and to be chemotactic for MDSC, are abundant in MDSC-derived exosomes. Bioassays reveal that MDSC-derived exosomes polarize macrophages towards a tumor-promoting type 2 phenotype, in addition to possessing S100A8/A9 chemotactic activity. These results suggest that some of the tumor-promoting functions of MDSC are implemented by MDSC-shed exosomes.
doi:10.1021/pr400879c
PMCID: PMC3946337  PMID: 24295599
extracellular vesicles; exosomes; myeloid-derived suppressor cells; chemotaxis; macrophages; proteomics; spectral counting; tumors; protein S100A8; immune suppression
10.  Differentiation of Cancer Cell Origin and Molecular Subtype by Plasma Membrane N-Glycan Profiling 
Journal of proteome research  2014;13(2):961-968.
In clinical settings, biopsies are routinely used to determine cancer type and grade based on tumor cell morphology, as determined via histochemical or immunohistochemical staining. Unfortunately, in a significant number of cases, traditional biopsy results are either inconclusive or do not provide full subtype differentiation, possibly leading to inefficient or ineffective treatment. Glycomic profiling of the cell membrane offers an alternate route towards cancer diagnosis. In this study, isomer-sensitive nano-LC/MS was used to directly obtain detailed profiles of the different N-glycan structures present on cancer cell membranes. Membrane N-glycans were extracted from cells representing various subtypes of breast, lung, cervical, ovarian, and lymphatic cancer. Chip-based porous graphitized carbon nano-LC/MS was used to separate, identify, and quantify the native N-glycans. Structure-sensitive N-glycan profiling identified hundreds of glycan peaks per cell line, including multiple isomers for most compositions. Hierarchical clusterings based on Pearson correlation coefficients were used to quickly compare and separate each cell line according to originating organ and disease subtype. Based simply on the relative abundances of broad glycan classes (e.g. high mannose, complex/hybrid fucosylated, complex/hybrid sialylated, etc.) most cell lines were readily differentiated. More closely-related cell lines were differentiated based on several-fold differences in the abundances of individual glycans. Based on characteristic N-glycan profiles, primary cancer origins and molecular subtypes could be distinguished. These results demonstrate that stark differences in cancer cell membrane glycosylation can be exploited to create an MS-based biopsy, with potential applications towards cancer diagnosis and direction of treatment.
doi:10.1021/pr400987f
PMCID: PMC3946297  PMID: 24303873
mass spectrometry; LC/MS; N-glycans; cell membrane; cancer; molecular subtype
11.  Discrimination between adenocarcinoma and normal pancreatic ductal fluid by proteomic and glycomic analysis 
Journal of proteome research  2013;13(2):395-407.
Sensitive and specific biomarkers for pancreatic cancer are currently unavailable. The high mortality associated with adenocarcinoma of the pancreatic epithelium justifies the broadest possible search for new biomarkers that can facilitate early detection or monitor treatment efficacy. Protein glycosylation is altered in many cancers, leading many to propose that glycoproteomic changes may provide suitable biomarkers. In order to assess this possibility for pancreatic cancer, we have performed an in-depth LC-MS/MS analysis of the proteome and MSn-based characterization of the N-linked glycome of a small set of pancreatic ductal fluid obtained from normal, pancreatitis, intraductal papillary mucinous neoplasm (IPMN), and pancreatic adenocarcinoma patients. Our results identify a set of seven proteins that were consistently increased in cancer ductal fluid compared to normal (AMYP, PRSS1, GP2-1, CCDC132, REG1A, REG1B, and REG3A) and one protein that was consistently decreased (LIPR2). These proteins are all directly or indirectly associated with the secretory pathway in normal pancreatic cells. Validation of these changes in abundance by Western blotting revealed increased REG protein glycoform diversity in cancer. Characterization of the total N-linked glycome of normal, IPMN, and adenocarcinoma ductal fluid clustered samples into three discrete groups based on the prevalence of 6 dominant glycans. Within each group, the profiles of less prevalent glycans were able to distinguish normal from cancer on this small set of samples. Our results emphasize that individual variation in protein glycosylation must be considered when assessing the value of a glycoproteomic marker, but also indicate that glycosylation diversity across human subjects can be reduced to simpler clusters of individuals whose N-linked glycans share structural features.
doi:10.1021/pr400422g
PMCID: PMC3946306  PMID: 24328148
Pancreatic cancer; Proteomics; Biomarker; N-linked glycan; Glycomics
12.  SILAC Peptide Ratio Calculator - A tool for SILAC Quantitation of Peptides and Posttranslational Modifications 
Journal of proteome research  2014;13(2):506-516.
This paper describes an algorithm to assist in relative quantitation of peptide post-translational modifications using Stable Isotope Labeling by Amino acids in Cell culture (SILAC). The described algorithm first determines the normalization factor and then calculates SILAC ratios for a list of target peptide masses using precursor ion abundances. Four yeast histone mutants were used to demonstrate the effectiveness of this approach for quantitation of peptide post-translational modifications changes. The details of the algorithm’s approach for normalization and peptide ratio calculation are described. The examples demonstrate the robustness of the approach as well as its utility to rapidly determine changes in peptide posttranslational modifications within a protein.
doi:10.1021/pr400675n
PMCID: PMC3953147  PMID: 24328097
Quantitative proteomics; global normalization factor; ratio/charge filter; peptide clustering; sliding time window
13.  Metabolomic profiling identifies biochemical pathways associated with castrate resistant prostate cancer 
Journal of proteome research  2013;13(2):1088-1100.
Despite recent developments in treatment strategies, castrate resistant prostate cancer (CRPC) is still the second leading cause of cancer associated mortality among American men, the biological underpinnings of which are not well understood. To this end, we measured levels of 150 metabolites and examined the rate of utilization of 184 metabolites in metastatic androgen dependent prostate cancer (AD) and CRPC cell lines using a combination of targeted mass spectrometry and metabolic phenotyping. Metabolic data were used to derive biochemical pathways that were enriched in CRPC, using Oncomine Concept Maps (OCM). The enriched pathways were then examined in-silico for their association with treatment failure (i.e., prostate specific antigen (PSA) recurrence or biochemical recurrence) using published clinically annotated gene expression data sets. Our results indicate that a total of 19 metabolites were altered in CRPC compared to AD cell lines. These altered metabolites mapped to a highly interconnected network of biochemical pathways that describe UDP glucuronosyltransferase (UGT) activity. We observed an association with time to treatment failure in an analysis employing genes restricted to this pathway in three independent gene expression data sets. In summary, our studies highlight the value of employing metabolomic strategies in cell lines to derive potentially clinically useful predictive tools.
doi:10.1021/pr401106h
PMCID: PMC3975657  PMID: 24359151
Prostate Cancer; Castrate Resistant Prostate Cancer; Metabolomics; Metabolic Phenotyping; Liquid-chromatography Mass Spectrometry; Oncomine Concept Map and Biochemical Recurrence
14.  Enhanced identification of zero-length chemical crosslinks using label-free quantitation and high-resolution fragment ion spectra 
Journal of proteome research  2014;13(2):898-914.
Chemical crosslinking coupled with mass spectrometry provides structural information that is useful for probing protein conformations and providing experimental support for molecular models. “Zero-length” crosslinks have greater value for these applications than longer crosslinks because they provide more stringent distance constraints. However, this method is less commonly utilized because it cannot take advantage of isotopic labels, MS-labile bonds, or enrichment tags to facilitate identification. In this study, we combined label-free precursor ion quantitation and targeted tandem mass spectrometry with a new software tool, Zero-length Crosslink Miner (ZXMiner), to form a multi-tiered analysis strategy. A major, critical objective was to simultaneously achieve very high accuracy with essentially no false positive crosslink identifications, while maintaining a good depth of analysis. Our strategy was optimized on several proteins with known crystal structures. Comparison of ZXMiner to several existing crosslink analysis software showed that other algorithms detected less true positive crosslinks and were far less accurate. Although prior use of zero-length crosslinking was typically restricted to small proteins, ZXMiner and the associated strategy enables facile analysis of very large protein complexes. This was demonstrated by identification of zero-length crosslinks using purified 526 kDa spectrin heterodimers and intact red cell membranes and membrane skeletons.
doi:10.1021/pr400953w
PMCID: PMC3975668  PMID: 24369724
chemical crosslinking; mass spectrometry; software
15.  Metabolomics Analysis Identifies D-Alanine-D-alanine Ligase as the Primary Lethal Target of D-cycloserine in Mycobacteria 
Journal of proteome research  2013;13(2):1065-1076.
D-cycloserine is an effective second line antibiotic used as a last resort to treat multi (MDR)- and extensively (XDR)- drug resistant strains of Mycobacterium tuberculosis. D-cycloserine interferes with the formation of peptidoglycan biosynthesis by competitive inhibition of Alanine racemase (Alr) and D-Alanine-D-alanine ligase (Ddl). Although, the two enzymes are known to be inhibited, the in vivo lethal target is still unknown. Our NMR metabolomics work has revealed that Ddl is the primary target of DCS, as cell growth is inhibited when the production of D-alanyl-D-alanine is halted. It is shown that inhibition of Alr may contribute indirectly by lowering the levels of D-alanine thus allowing DCS to outcompete D-alanine for Ddl binding. The NMR data also supports the possibility of a transamination reaction to produce D-alanine from pyruvate and glutamate, thereby bypassing Alr inhibition. Furthermore, the inhibition of peptidoglycan synthesis results in a cascading effect on cellular metabolism as there is a shift toward the catabolic routes to compensate for accumulation of peptidoglycan precursors.
doi:10.1021/pr4010579
PMCID: PMC3975674  PMID: 24303782
Tuberculosis; NMR metabolomics; mycobacteria; D-cycloserine
16.  Fast and accurate database searches with MS-GF+Percolator 
Journal of proteome research  2013;13(2):890-897.
One can interpret fragmentation spectra stemming from peptides in mass spectrometry-based proteomics experiments using so called database search engines. Frequently, one also runs post-processors such as Percolator to assess the confidence, infer unique peptides and increase the number of identifications. A recent search engine, MS-GF+, has shown promising results, due to a new and efficient scoring algorithm. However, MS-GF+ provides few statistical estimates about the peptide-spectrum matches, hence limiting the biological interpretation. Here, we enabled Percolator-processing for MS-GF+ output, and observed an increased number of identified peptides for a wide variety of datasets. In addition, Percolator directly reports p values and false discovery rate estimates, such as q values and posterior error probabilities, for peptide-spectrum matches, peptides and proteins, functions that are useful for the whole proteomics community.
doi:10.1021/pr400937n
PMCID: PMC3975676  PMID: 24344789
17.  Cigarette smoke induces distinct chromatin histone modifications in lung cells: implication in pathogenesis of COPD and lung cancer 
Journal of proteome research  2013;13(2):982-996.
Cigarette smoke (CS)-mediated oxidative stress induces several signaling cascades, including kinases, which results in chromatin modifications (histone acetylation/deacetylation and histone methylation/demethylation). We have previously reported that CS induces chromatin remodeling in pro-inflammatory gene promoters; however, the underlying site-specific histone marks formed in histones H3 and H4 during CS exposure in lungs in vivo and in lung cells in vitro, which can either drive gene expression or repression are not known. We hypothesize that CS exposure in mouse and human bronchial epithelial cells (H292) can cause site-specific posttranslational histone modifications (PTMs) that may play an important role in the pathogenesis of CS-induced chronic lung diseases. We used a bottom-up mass spectrometry approach to identify some potentially novel histone marks, including acetylation, mono-methylation and di-methylation in specific lysine and arginine residues of histones H3 and H4 in mouse lungs and H292 cells. We found that CS-induced distinct posttranslational histone modification patterns in histone H3 and histone H4 in lung cells, which may be considered as usable biomarkers for CS-induced chronic lung diseases. These identified histone marks (histone H3 and histone H4) may play an important role in epigenetic state during the pathogenesis of smoking-induced chronic lung diseases, such as chronic obstructive pulmonary disease and lung cancer.
doi:10.1021/pr400998n
PMCID: PMC3975679  PMID: 24283195
Oxidants; chromatin; lung; acetylation; methylation; chronic obstructive pulmonary disease; mass spectrometry; lung cancer
18.  The Conserved Sequence Repeats of IQGAP1 mediate binding to Ezrin 
Journal of proteome research  2013;13(2):1156-1166.
Mammalian IQGAP proteins all feature multiple ~50 amino acid sequence repeats near their N-termini and little is known about the function of these “Repeats”. We have expressed and purified the Repeats from human IQGAP1 in order to identify binding partners. We used mass spectrometry to identify 42 mouse kidney proteins that associate with the IQGAP1 Repeats including the ERM proteins ezrin, radixin and moesin. ERM proteins have an N-terminal FERM domain (four point one, ezrin, radixin, moesin) through which they bind to protein targets and phosphatidylinositol 4,5-bisphosphate (PIP2), and a C-terminal actin-binding domain, and function to link the actin cytoskeleton to distinct locations on the cell cortex. Isothermal titration calorimetry (ITC) reveals that the IQGAP1 Repeats directly bind to the ezrin FERM domain while no binding is seen for full-length “autoinhibited” ezrin or a version of full-length ezrin intended to mimic the activated protein. ITC also indicates that the ezrin FERM domain binds to the Repeats from IQGAP2 but not the Repeats from IQGAP3. We conclude that IQGAP1 and IQGAP2 are positioned at the cell cortex by ERM proteins. We propose that the IQGAP3 Repeats may likewise bind to FERM domains signaling purposes.
doi:10.1021/pr400787p
PMCID: PMC3975686  PMID: 24294828
IQGAP1; Repeats; ezrin; FERM domain
19.  A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum 
Journal of proteome research  2013;13(2):875-882.
Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase LC separations to fractionate and select target fractions for follow-on LC-SRM analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and LOQ values of ~130 pg/mL in serum and ~10 pg per 100 μg total protein mass in urine, respectively. A good correlation (R2 = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and ELISA. Based on an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between non-cancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.
doi:10.1021/pr400912c
PMCID: PMC3975687  PMID: 24251762
AGR2; PSA; prostate cancer; PRISM-SRM; human urine; human serum
20.  Proteomics Reveals Age-related Differences in the Host Immune Response to Sepsis 
Journal of proteome research  2013;13(2):422-432.
Sepsis is commonly caused by community-acquired pneumonia (CAP) and may develop into severe sepsis, characterized by multiple organ failure. The risk of severe sepsis among CAP patients and subsequent mortality increases sharply after the age of 65. The molecular mechanisms associated with this age-related risk are not fully understood. To better understand factors involved with increased incidence and mortality of severe sepsis in the elderly, we used a nested case-control study of patients enrolled in a multicenter observational cohort of 2,320 participants with CAP. We identified a total of 39 CAP patients 50-65 and 70-85 years old who did or did not develop severe sepsis. Plasma samples were obtained on presentation to the emergency department and prior to therapeutic interventions. A semi-quantitative plasma proteomics workflow was applied which incorporated tandem immunoaffinity depletion, iTRAQ labeling, strong cation exchange fractionation, and nanoflow-liquid chromatography coupled to high resolution mass spectrometry. In total, 772 proteins were identified, of which, 58 proteins exhibit statistically significant differences in expression levels amongst patients with severe sepsis as a function of age. Differentially-expressed proteins are involved in pathways such as acute phase response, coagulation signaling, atherosclerosis signaling, lipid metabolism, and production of nitric oxide and reactive oxygen species. This study provides insight into factors that may explain age-related differences in incidence of severe sepsis in the elderly.
doi:10.1021/pr400814s
PMCID: PMC3976438  PMID: 24266763
sepsis; severe sepsis; proteomics; CAP; plasma; aging; immunosenescence; pneumonia
21.  Accurate Identification of Deamidated Peptides in Global Proteomics using a Quadrupole Orbitrap Mass Spectrometer 
Journal of proteome research  2013;13(2):777-785.
Deamidation of asparagine and glutamine residues is a common post-translational modification. Researchers often rely on mass spectrometric based proteomic techniques for the identification of these post-translational sites. Mass spectral analysis of deamidated peptides is complicated and often misassigned due to overlapping 13C peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by 19.34 mDa. For proper assignment it is inherently important to use a mass spectrometer with high mass measurement accuracy and high resolving power. Herein, mouse brain tissue lysate was prepared using filter-aided sample preparation (FASP) method and Stage Tip fractionation followed by analysis on a nanoLC coupled to a quadrupole orbitrap (Q-Exactive) mass spectrometer to accurately identify more than 5400 proteins. Mass spectral data was processed using MASCOT and ProteoIQ for accurate identification of peptides and proteins. MASCOT search values for precursor and MS/MS mass tolerances were investigated, and it was determined data searched with greater than 5 ppm precursor mass tolerance resulted in the misassignment of deamidated peptides. Peptides that were identified with a mass measurement accuracy of ± 5 ppm were correctly assigned.
doi:10.1021/pr400848n
PMCID: PMC3976439  PMID: 24289162
22.  Crossword: A fully automated algorithm for the segmentation and quality control of protein microarray images 
Journal of proteome research  2014;13(2):362-371.
Biological assays formatted as microarrays have become a critical tool for the generation of the comprehensive datasets required for systems-level understanding of biological processes. Manual annotation of data extracted from images of microarrays, however, remains a significant bottleneck, particularly for protein microarrays due to the sensitivity of this technology to weak artifact signal. In order to automate the extraction and curation of data from protein microarrays, we describe an algorithm called Crossword that logically combines information from multiple approaches for segmenting microarrays and a novel method for removing artifacts. Artifact removal is accomplished by segregating structured pixels from the background noise using iterative clustering and pixel connectivity. Correlation of the location of structured pixels across image channels is used to identify and remove artifact pixels from the image prior to data extraction. This component improves the accuracy of datasets while reducing the requirement for time-consuming visual inspection of the data. Crossword enables a fully automated protocol that is robust to significant spatial and intensity aberrations. Overall, the average amount of user intervention is reduced by an order of magnitude and the data quality is increased through artifact removal and reduced user variability. The increase in throughput should aid the further implementation of microarray technologies in clinical studies.
doi:10.1021/pr401167h
PMCID: PMC3979532  PMID: 24417579
image segmentation; artifact removal; image quality control; automated image processing; pixel clustering; pixel connectivity
23.  Glycomic Analysis of High Density Lipoprotein (HDL) Shows a Highly Sialylated Particle 
Journal of proteome research  2014;13(2):681-691.
Many of the functional proteins and lipids in HDL particles are potentially glycosylated yet very little is known about the glycoconjugates of HDL. In this study, HDL was isolated from plasma by sequential micro-ultracentrifugation, followed by glycoprotein and glycolipid analysis. N-glycans, glycopeptides, and gangliosides were extracted and purified followed by analysis with nano-HPLC-Chip Q-TOF MS and MS/MS. HDL particles were found to be highly sialylated. Most of the N-glycans (~90%) from HDL glycoproteins were sialylated with one or two neuraminic acids (Neu5Ac). The most abundant N-glycan was a biantennary complex type glycan with two sialic acids (Hexose5HexNAc4Neu5Ac2), and was found in multiple glycoproteins using site-specific glycosylation analysis. The observed O-glycans were all sialylated and most contained a core 1 structure with two Neu5Acs, including those that were associated with apolipoprotein CIII (ApoC-III) and fetuin A. GM3 (monosialoganglioside, NeuAc2-3Gal1-4Glc-Cer) and GD3 (disialoganglioside, NeuAc2-8NeuAc2-3Gal1-4Glc-Cer) were the major gangliosides in HDL. A 60% GM3 and 40% GD3 distribution was observed. Both GM3 and GD3 were composed of heterogeneous ceramide lipid tails, including d18:1/16:0 and d18:1/23:0. This report describes for the first time a glycomic approach for analyzing HDL, highlighting that HDL are highly sialylated particles.
doi:10.1021/pr4012393
PMCID: PMC3975653  PMID: 24417605
HDL; glycomics; glycoproteomics; gangliosides; sialylation; mass spectrometry
24.  A Large Scale, Ion Current based Proteomics Investigation of Bronchoalveolar Lavage Fluid in Chronic Obstructive Pulmonary Disease Patients 
Journal of proteome research  2013;13(2):627-639.
Proteomic analysis of bronchoalveolarBlavageBfluid (BALF) in chronic obstructive pulmonary disease (COPD) patients may provide new biomarkers and deeper understanding of the disease mechanisms but remains challenging. Here we describe an ionBcurrentBbased strategy for comparative analysis of BALF proteomes from patients with moderate and stable COPD vs. healthy controls. The strategy includes an efficient preparation procedure providing quantitative recovery and a nanoBLC/MS analysis with a long, heated column. Under optimized conditions, high efficiency and reproducibility were achieved for each step, enabling a “20Bplex” comparison of clinical subjects (n=10/group). Without depletion/fractionation, a total of 423 unique protein groups were quantified under stringent criteria with at least two quantifiable peptides. SeventyBsix proteins were determined as significantlyBaltered in COPD, which represent a diversity of biological processes such as alcohol metabolic process, gluconeogenesis/glycolysis, inflammatory response, proteolysis, and oxidation reduction. Interestingly, altered alcohol metabolism responding to oxidant stress is a novel observation in COPD. The prominently elevated key enzymes involved in alcohol metabolism (e.g. ADH1B, ALDH2&ALDH3A1) may provide a reasonable explanation for a bewildering observation in COPD patients known for decades: the underestimation of the blood alcohol concentrations through breath tests. These discoveries could provide new insights for identifying novel biomarkers and pathological mediators in clinical studies.
doi:10.1021/pr4007602
PMCID: PMC4073647  PMID: 24188068
Biomarker Discovery; Bronchoalveolar Lavage Fluid; Chronic Obstructive Pulmonary Disease; Peptide Extracted Ion Current
25.  Untargeted LC-MS Metabolomics of Bronchoalveolar Lavage Fluid Differentiates Acute Respiratory Distress Syndrome from Health 
Journal of proteome research  2013;13(2):640-649.
Acute respiratory distress syndrome (ARDS) remains a significant hazard to human health and is clinically challenging because there are no prognostic biomarkers and no effective pharmacotherapy. The lung compartment metabolome may detail the status of the local environment that could be useful in ARDS biomarker discovery and the identification of drug target opportunities. However, neither the utility of bronchoalveolar lavage fluid (BALF) as a biofluid for metabolomics nor the optimal analytical platform for metabolite identification are established. To address this, we undertook a study to compare metabolites in BALF samples from patients with ARDS and healthy controls using a newly developed liquid chromatography (LC)-mass spectroscopy (MS) platform for untargeted metabolomics. Following initial testing of three different high performance liquid chromatography (HPLC) columns, we determined that reversed phase (RP)-LC and hydrophilic interaction chromatography (HILIC), were the most informative chromatographic methods because they yielded the most and highest quality data. Following confirmation of metabolite identification, statistical analysis resulted in 37 differentiating metabolites in the BALF of ARDS compared with health across both analytical platforms. Pathway analysis revealed networks associated with amino acid metabolism, glycolysis and gluconeogenesis, fatty acid biosynthesis, phospholipids and purine metabolism in the ARDS BALF. The complementary analytical platforms of RPLC and HILIC-LC generated informative, insightful metabolomics data of the ARDS lung environment.
doi:10.1021/pr4007624
PMCID: PMC4068805  PMID: 24289193
biomarkers; critical illness; metabolomics; lung injury; bioinformatics; phospholipids; lactate; xanthine oxidase; hippurate; pharmacotherapy

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