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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.  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
3.  Proteomic approaches for site-specific O-GlcNAcylation analysis 
Bioanalysis  2014;6(19):2571-2580.
O -GlcNAcylation is a dynamic protein post-translational modification of serine or threonine residues by an O-linked monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation was discovered three decades ago and its significance has been implicated in several disease states, such as metabolic diseases, cancer and neurological diseases. Yet it remains technically challenging to characterize comprehensively and quantitatively because of its low abundance, low stoichiometry and extremely labile nature under conventional collision-induced dissociation tandem MS conditions. Herein, we review the recent advances addressing these challenges in developing proteomic approaches for site-specific O-GlcNAcylation analysis, including specific enrichment of O-GlcNAc peptides/proteins, unambiguous site-determination of O-GlcNAc modification and quantitative analysis of O-GlcNAcylation.
doi:10.4155/bio.14.239
PMCID: PMC4275047  PMID: 25411699
4.  HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa 
PLoS Genetics  2014;10(11):e1004783.
Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication.
Author Summary
Cell fusion between genetically identical cells of the fungus Neurospora crassa occurs when germinating asexual cells (conidia) sense each other's proximity and redirect their growth. Chemotropic growth is dependent upon the assembly of a MAPK cascade (NRC-1/MEK-2/MAK-2) at the cell cortex (conidial anastomosis tubes; CATs), followed by disassembly over an ∼8 min cycle. A second protein required for fusion, SO, also assembles and disassembles at CAT tips during chemotropic growth, but with perfectly opposite dynamics to the MAK-2 complex. This process of germling chemotropism, oscillation and cell fusion is regulated by many genes and is poorly understood. Via a phosphoproteomics approach, we identify HAM-5, which functions as a scaffold for the MAK-2 signal transduction complex. HAM-5 is required for assembly/disassembly and oscillation of the MAK-2 complex during chemotropic growth. Our data supports a model whereby regulated modification of HAM-5 controls the disassembly of the MAK-2 MAPK complex and is essential for modulating the tempo of oscillation during chemotropic interactions.
doi:10.1371/journal.pgen.1004783
PMCID: PMC4238974  PMID: 25412208
5.  Antibody-independent targeted quantification of TMPRSS2-ERG fusion protein products in prostate cancer 
Molecular oncology  2014;8(7):1169-1180.
Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. Studies of TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies suitable for quantitative studies. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays provided confident detection of 6 unique ERG peptides in both TMPRSS2-ERG positive cell lines and tissues, but not in cell lines or tissues lacking the TMPRSS2-ERG rearrangement, clearly indicating that ERG protein expression is significantly increased in the presence of the TMPRSS2-ERG gene fusion. Significantly, our results provide evidence that two distinct ERG protein isoforms are simultaneously expressed in TMPRSS2-ERG positive samples as evidenced by the concomitant detection of two mutually exclusive peptides in two patient tumors and in the VCaP prostate cancer cell line. Three peptides, shared across almost all fusion protein products, were determined to be the most abundant peptides, providing “signature” peptides for detection of ERG over-expression resulting from TMPRSS2-ERG gene fusion. The PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products in prostate cancer.
doi:10.1016/j.molonc.2014.02.004
PMCID: PMC4183720  PMID: 25266362
TMPRSS2-ERG gene fusion; ERG protein isoform; PRISM-SRM; Targeted quantification; Prostate cancer
6.  Expediting SRM assay development for large-scale targeted proteomics experiments 
Journal of proteome research  2014;13(10):4479-4487.
Due to their high sensitivity and specificity, selected reaction monitoring (SRM) based targeted proteomics has become increasingly popular for biological and translational applications. Selection of optimal transitions and optimization of collision energy (CE) are important assay development steps for achieving sensitive detection and accurate quantification; however, these steps can be labor-intensive, especially, for large-scale applications. Herein, we explored several options for accelerating SRM assay development evaluated in the context of a relatively large set of 215 synthetic peptide targets. We first showed that HCD fragmentation is very similar to CID in triple quadrupole (QQQ) instrumentation, and by selection of the top six y fragment ions from HCD spectra, >86% of the top transitions optimized from direct infusion on QQQ instrument are covered. We also demonstrated that the CE calculated by existing prediction tools was less accurate for 3+ precursors, and a significant increase in intensity for transitions could be obtained using a new CE prediction equation constructed from the present experimental data. Overall, our study illustrated the feasibility of expediting the development of larger numbers of high-sensitivity SRM assays through automation of transition selection and accurate prediction of optimal CE to improve both SRM throughput and measurement quality.
doi:10.1021/pr500500d
PMCID: PMC4184450  PMID: 25145539
SRM; MRM; HCD; QQQ; transition selection; optimization; CE prediction; targeted quantification
7.  Long-gradient separations coupled with selected reaction monitoring for highly sensitive, large scale targeted protein quantification in a single analysis 
Analytical chemistry  2013;85(19):10.1021/ac402105s.
Long-gradient separations coupled to tandem MS were recently demonstrated to provide a deep proteome coverage for global proteomics; however, such long-gradient separations have not been explored for targeted proteomics. Herein, we investigate the potential performance of the long-gradient separations coupled with selected reaction monitoring (LG-SRM) for targeted protein quantification. Direct comparison of LG-SRM (5 h gradient) and conventional LC-SRM (45 min gradient) showed that the long-gradient separations significantly reduced background interference levels and provided an 8- to 100-fold improvement in LOQ for target proteins in human female serum. Based on at least one surrogate peptide per protein, an LOQ of 10 ng/mL was achieved for the two spiked proteins in non-depleted human serum. The LG-SRM detection of seven out of eight endogenous plasma proteins expressed at ng/mL or sub-ng/mL levels in clinical patient sera was also demonstrated. A correlation coefficient of >0.99 was observed for the results of LG-SRM and ELISA measurements for prostate-specific antigen (PSA) in selected patient sera. Further enhancement of LG-SRM sensitivity was achieved by applying front-end IgY14 immunoaffinity depletion. Besides improved sensitivity, LG-SRM potentially offers much higher multiplexing capacity than conventional LC-SRM due to an increase in average peak widths (~3-fold) for a 300-min gradient compared to a 45-min gradient. Therefore, LG-SRM holds great potential for bridging the gap between global and targeted proteomics due to its advantages in both sensitivity and multiplexing capacity.
doi:10.1021/ac402105s
PMCID: PMC3839867  PMID: 24004026
long-gradient; targeted quantification; low-abundance protein; human serum; sensitivity; reproducibility
8.  Trauma-associated Human Neutrophil Alterations Revealed by Comparative Proteomics Profiling 
Proteomics. Clinical applications  2013;7(0):10.1002/prca.201200109.
PURPOSE
Polymorphonuclear neutrophils (PMNs) play an important role in mediating the innate immune response after severe traumatic injury; however, the cellular proteome response to traumatic condition is still largely unknown.
EXPERIMENTAL DESIGN
We applied 2D-LC-MS/MS based shotgun proteomics to perform comparative proteome profiling of human PMNs from severe trauma patients and healthy controls.
RESULTS
A total of 197 out of ~2500 proteins (being identified with at least two peptides) were observed with significant abundance changes following the injury. The proteomics data were further compared with transcriptomics data for the same genes obtained from an independent patient cohort. The comparison showed that the protein abundance changes for the majority of proteins were consistent with the mRNA abundance changes in terms of directions of changes. Moreover, increased protein secretion was suggested as one of the mechanisms contributing to the observed discrepancy between protein and mRNA abundance changes. Functional analyses of the altered proteins showed that many of these proteins were involved in immune response, protein biosynthesis, protein transport, NRF2-mediated oxidative stress response, the ubiquitin-proteasome system, and apoptosis pathways.
CONCLUSIONS AND CLINICAL RELEVANCE
Our data suggest increased neutrophil activation and inhibited neutrophil apoptosis in response to trauma. The study not only reveals an overall picture of functional neutrophil response to trauma at the proteome level, but also provides a rich proteomics data resource of trauma-associated changes in the neutrophil that will be valuable for further studies of the functions of individual proteins in PMNs.
doi:10.1002/prca.201200109
PMCID: PMC3737403  PMID: 23589343
human neutrophil; LC-MS/MS; Proteomics; Trauma; Genomics
9.  GlyQ-IQ: Glycomics Quintavariate-Informed Quantification with High-Performance Computing and GlycoGrid 4D Visualization 
Analytical chemistry  2014;86(13):6268-6276.
Glycomics quintavariate-informed quantification (GlyQ-IQ) is a biologically guided glycomics analysis tool for identifying N-glycans in liquid chromatography–mass spectrometry (LC–MS) data. Glycomics LC–MS data sets have convoluted extracted ion chromatograms that are challenging to deconvolve with software. LC deconvolution into constituent pieces is critical in glycomics data sets because chromatographic peaks correspond to different intact glycan structural isomers. The biological targeted analysis approach offers several key advantages to traditional LC–MS data processing. A priori glycan information about the individual target's elemental composition allows for improved sensitivity by utilizing the exact isotope profile information to focus chromatogram generation and LC peak fitting on the isotopic species having the highest intensity. Glycan target annotation utilizes glycan family relationships and in source fragmentation in addition to high specificity feature LC–MS detection to improve the specificity of the analysis. The GlyQ-IQ software is developed in this work and was used to profile N-glycan compositions from human serum LC–MS data sets. A case study is presented to demonstrate how GlyQ-IQ identifies and removes confounding chromatographic peaks from high mannose glycan isomers from human blood serum. In addition, GlyQ-IQ was used to generate a broad human serum N-glycan profile from a high resolution nanoelectrospray-liquid chromatography–tandem mass spectrometry (nESI-LC–MS/MS) data set. A total of 156 glycan compositions and 640 glycan isomers were detected from a single sample. Over 99% of the GlyQ-IQ glycan-feature assignments passed manual validation and are backed with high-resolution mass spectra.
doi:10.1021/ac501492f
PMCID: PMC4082388  PMID: 24881670
10.  Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing 
Analytical chemistry  2013;85(14):6826-6832.
A resin-assisted enrichment method has been developed for specific isolation of protein N-terminal peptides to facilitate LC-MS/MS characterization of proteolytic processing, a major form of posttranslational modifications. In this method, protein thiols are blocked by reduction and alkylation, and protein lysine residues are converted to homoarginines. Protein N-termini are selectively converted to reactive thiol groups, and the thiol-containing N-terminal peptides are then captured by a thiol-affinity resin with high specificity (>97%). The efficiencies of these sequential reactions were demonstrated to be nearly quantitative. The resin-assisted N-terminal peptide enrichment approach was initially applied to a cell lysate of the filamentous fungus Aspergillus niger. Subsequent C-MS/MS analyses resulted in the identification of 1672 unique protein N-termini or proteolytic cleavage sites from 690 unique proteins.
doi:10.1021/ac401000q
PMCID: PMC3782086  PMID: 23772796
proteolytic processing; N-terminal peptides; enrichment; Aspergillus niger
11.  Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion 
Journal of proteome research  2013;12(7):3353-3361.
We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50–100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ~12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successful quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies.
doi:10.1021/pr400178v
PMCID: PMC3733379  PMID: 23763644
SRM; PRISM; targeted quantification; low-abundance protein; human serum; sensitivity; reproducibility
12.  Determination of burn patient outcome by large-scale quantitative discovery proteomics 
Critical care medicine  2013;41(6):1421-1434.
Objective
Emerging proteomics techniques can be used to establish proteomic outcome signatures and to identify candidate biomarkers for survival following traumatic injury. We applied high-resolution liquid chromatography-mass spectrometry (LC-MS) and multiplex cytokine analysis to profile the plasma proteome of survivors and non-survivors of massive burn injury to determine the proteomic survival signature following a major burn injury.
Design
Proteomic discovery study.
Setting
Five burn hospitals across the U.S.
Patients
Thirty-two burn patients (16 non-survivors and 16 survivors), 19–89 years of age, were admitted within 96 h of injury to the participating hospitals with burns covering >20% of the total body surface area and required at least one surgical intervention.
Interventions
None.
Measurements and Main Results
We found differences in circulating levels of 43 proteins involved in the acute phase response, hepatic signaling, the complement cascade, inflammation, and insulin resistance. Thirty-two of the proteins identified were not previously known to play a role in the response to burn. IL-4, IL-8, GM-CSF, MCP-1, and β2-microglobulin correlated well with survival and may serve as clinical biomarkers.
Conclusions
These results demonstrate the utility of these techniques for establishing proteomic survival signatures and for use as a discovery tool to identify candidate biomarkers for survival. This is the first clinical application of a high-throughput, large-scale LC-MS-based quantitative plasma proteomic approach for biomarker discovery for the prediction of patient outcome following burn, trauma or critical illness.
doi:10.1097/CCM.0b013e31827c072e
PMCID: PMC3660437  PMID: 23507713
burn; inflammation; proteomic profiling; plasma proteins; LC-MS; biomarker
13.  Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications 
Nature protocols  2013;9(1):64-75.
Reversible modifications of cysteine thiols play a significant role in redox signaling and regulation. A number of reversible redox modifications, including disulfide formation, S-nitrosylation, and S-glutathionylation, have been recognized for their significance in various physiological and pathological processes. Here we describe a procedure for the enrichment of peptides containing reversible cysteine modifications. Starting with tissue or cell lysate samples, all of the unmodified free thiols are blocked using N-ethylmaleimide (NEM). This is followed by the selective reduction of those cysteines bearing the reversible modification(s) of interest. The reduction is achieved by using different reducing reagents that react specifically with each type of cysteine modification (e.g., ascorbate for S-nitrosylation). This protocol serves as a general approach for enrichment of thiol-containing proteins or peptides derived from reversibly modified proteins. The approach utilizes a commercially available thiol-affinity resin (Thiopropyl Sepharose 6B) to directly capture free thiol-containing proteins through a disulfide exchange reaction followed by on-resin protein digestion and multiplexed isobaric labeling to facilitate LC–MS/MS based quantitative site-specific analysis of cysteine-based reversible modifications. The overall approach requires a simpler workflow with increased specificity compared to the commonly used biotinylation-based assays. The procedure for selective enrichment and analyses of S-nitrosylation and the level of total reversible cysteine modifications (or total oxidation) is presented to demonstrate the utility of this general strategy. The entire protocol requires approximately 3 days for sample processing with an additional day for LC-MS/MS and data analysis.
doi:10.1038/nprot.2013.161
PMCID: PMC4038159  PMID: 24336471
Mass spectrometry; post-translational modification; cysteine modification; redox modification; cysteine derivatisation; on-resin digestion; on-resin reaction; thiol enrichment; S-nitrosylation; reversibly oxidized cysteines; S-glutathionylation; S-acylation; iTRAQ; isobaric tags for relative and absolute quantification; tandem mass tags; TMT; MASIC
14.  Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry 
S-nitrosylation, the formation of S-nitrosothiol (SNO), is an important reversible thiol oxidation event that has been increasingly recognized for its role in cell signaling. Although many proteins susceptible to S-nitrosylation have been reported, site-specific identification of physiologically relevant SNO modifications remains an analytical challenge because of the low abundance and labile nature of this modification. Herein we present further improvement and optimization of the recently reported resin-assisted cysteinyl peptide enrichment protocol for SNO identification and its application to mouse skeletal muscle to identify specific cysteine sites sensitive to S-nitrosylation by a quantitative reactivity profiling strategy. Our results indicate that the protein- and peptide-level enrichment protocols provide comparable specificity and coverage of SNO-peptide identifications. S-nitrosylation reactivity profiling was performed by quantitatively comparing the site-specific SNO modification levels in samples treated with S-nitrosoglutathione, an NO donor, at two different concentrations (i.e., 10 and 100 μM). The reactivity profiling experiments led to the identification of 488 SNO-modified sites from 197 proteins with specificity of ~95% at the unique peptide level, i.e., ~95% of enriched peptides contain cysteine residues as the originally SNO-modified sites. Among these sites, 281 from 145 proteins were considered more sensitive to S-nitrosylation based on the ratios of observed SNO levels between the two treatments. These SNO-sensitive sites are more likely to be physiologically relevant. Many of the SNO-sensitive proteins are localized in mitochondria, contractile fiber, and actin cytoskeleton, suggesting the susceptibility of these subcellular compartments to redox regulation. Moreover, these observed SNO-sensitive proteins are primarily involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis/gluconeogenesis, glutathione metabolism, and fatty acid metabolism, suggesting the importance of redox regulation in muscle metabolism and insulin action.
doi:10.1016/j.freeradbiomed.2012.12.010
PMCID: PMC3771501  PMID: 23277143
S-nitrosylation; Redox regulation; Chemical enrichment; Mouse muscle; Proteomics; LC–MS/MS; Free radicals
15.  Cytomegalovirus pp65 limits dissemination but is dispensable for persistence 
The Journal of Clinical Investigation  2014;124(5):1928-1944.
The most abundantly produced virion protein in human cytomegalovirus (HCMV) is the immunodominant phosphoprotein 65 (pp65), which is frequently included in CMV vaccines. Although it is nonessential for in vitro CMV growth, pp65 displays immunomodulatory functions that support a potential role in primary and/or persistent infection. To determine the contribution of pp65 to CMV infection and immunity, we generated a rhesus CMV lacking both pp65 orthologs (RhCMVΔpp65ab). While deletion of pp65ab slightly reduced growth in vitro and increased defective particle formation, the protein composition of secreted virions was largely unchanged. Interestingly, pp65 was not required for primary and persistent infection in animals. Immune responses induced by RhCMVΔpp65ab did not prevent reinfection with rhesus CMV; however, reinfection with RhCMVΔUS2-11, which lacks viral-encoded MHC-I antigen presentation inhibitors, was prevented. Unexpectedly, induction of pp65b-specific T cells alone did not protect against RhCMVΔUS2-11 challenge, suggesting that T cells targeting multiple CMV antigens are required for protection. However, pp65-specific immunity was crucial for controlling viral dissemination during primary infection, as indicated by the marked increase of RhCMVΔpp65ab genome copies in CMV-naive, but not CMV-immune, animals. Our data provide rationale for inclusion of pp65 into CMV vaccines but also demonstrate that pp65-induced T cell responses alone do not recapitulate the protective effect of natural infection.
doi:10.1172/JCI67420
PMCID: PMC4002596  PMID: 24691437
16.  Steps: A Grid Search Methodology for Optimized Peptide Identification Filtering of MS/MS Database Search Results 
Proteomics  2013;13(5):766-770.
For bottom-up proteomics there are a wide variety of database searching algorithms in use for matching peptide sequences to tandem MS spectra. Likewise, there are numerous strategies being employed to produce a confident list of peptide identifications from the different search algorithm outputs. Here we introduce a grid search approach for determining optimal database filtering criteria in shotgun proteomics data analyses that is easily adaptable to any search. Systematic Trial and Error Parameter Selection – referred to as STEPS – utilizes user-defined parameter ranges to test a wide array of parameter combinations to arrive at an optimal “parameter set” for data filtering, thus maximizing confident identifications. The benefits of this approach in terms of numbers of true positive identifications are demonstrated using datasets derived from immunoaffinity-depleted blood serum and a bacterial cell lysate, two common proteomics sample types.
doi:10.1002/pmic.201200096
PMCID: PMC3676282  PMID: 23303698
17.  Metabolic Reprogramming during Purine Stress in the Protozoan Pathogen Leishmania donovani 
PLoS Pathogens  2014;10(2):e1003938.
The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6–48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.
Author Summary
Leishmania, the cause of a deadly spectrum of diseases in humans, surmounts a number of environmental challenges, including changes in the availability of salvageable nutrients, to successfully colonize its host. Adaptation to environmental stress is clearly of significance in parasite biology, but the underlying mechanisms are not well understood. To simulate the response to periodic nutrient scarcity in vivo, we have induced purine starvation in vitro. Purines are essential for growth and viability, and serve as the major energy currency of cells. Leishmania cannot synthesize purines and must salvage them from the surroundings. Extracellular purine depletion in culture induces a robust survival response in Leishmania, whereby growth arrests, but parasites persist for months. To profile the events that enable endurance of purine starvation, we used shotgun proteomics. Our data suggest that purine starvation induces extensive proteome remodeling, tailored to enhance purine capture and recycling, reduce energy expenditures, and maintain viability of the metabolically active, non-dividing population. Through global and targeted approaches, we reveal that proteome remodeling is multifaceted, and occurs through an array of responses at the mRNA, translational, and post-translational level. Our data provide one of the most inclusive views of adaptation to microenvironmental stress in Leishmania.
doi:10.1371/journal.ppat.1003938
PMCID: PMC3937319  PMID: 24586154
18.  Optimization for peptide sample preparation for urine peptidomics 
Clinical proteomics  2014;11(1):7.
Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput ‘peptidomics’ methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.
doi:10.1186/1559-0275-11-7
PMCID: PMC3944950  PMID: 24568099
Urine; Biomarker; Peptidomics; Biomarker discovery; Proteomics; Transplantation
19.  In-Source Fragmentation and the Sources of Partially Tryptic Peptides in Shotgun Proteomics 
Journal of proteome research  2013;12(2):910-916.
Partially tryptic peptides are often identified in shotgun proteomics using trypsin as the proteolytic enzyme; however, their sources have been controversial. Herein we investigate the impact of in-source fragmentation on shotgun proteomics profiling of three biological samples: a standard protein mixture, a mouse brain tissue homogenate, and mouse plasma. Since the in-source fragments of peptide ions have the same LC elution time as its parental peptide, partially tryptic peptide ions from in-source fragmentation can be distinguished from the other partially tryptic peptides based on their elution time differences from those computationally predicted data. The percentage of partially tryptic peptide identifications resulting from in-source fragmentation in a standard protein digest was observed to be ~60 %. In more complex mouse brain or plasma samples, in-source fragmentation contributed to a less degree of 1–3 % of all identified peptides due to the limit dynamic range of LC-MS/MS measurements. The other major source of partially tryptic peptides in complex biological samples is presumably proteolytic cleavage by endogenous proteases in the samples. Our work also provides a method to identify such proteolytic-derived partially tryptic peptides due to endogenous proteases in the samples by removing in-source fragmentation artifacts from the identified peptides.
doi:10.1021/pr300955f
PMCID: PMC3584699  PMID: 23268687
in-source fragmentation; partially tryptic; trypsin specificity; predicted elution time
20.  Phosphate-Containing Polyethylene Glycol Polymers Prevent Lethal Sepsis by Multidrug-Resistant Pathogens 
Antibiotic resistance among highly pathogenic strains of bacteria and fungi is a growing concern in the face of the ability to sustain life during critical illness with advancing medical interventions. The longer patients remain critically ill, the more likely they are to become colonized by multidrug-resistant (MDR) pathogens. The human gastrointestinal tract is the primary site of colonization of many MDR pathogens and is a major source of life-threatening infections due to these microorganisms. Eradication measures to sterilize the gut are difficult if not impossible and carry the risk of further antibiotic resistance. Here, we present a strategy to contain rather than eliminate MDR pathogens by using an agent that interferes with the ability of colonizing pathogens to express virulence in response to host-derived and local environmental factors. The antivirulence agent is a phosphorylated triblock high-molecular-weight polymer (here termed Pi-PEG 15–20) that exploits the known properties of phosphate (Pi) and polyethylene glycol 15-20 (PEG 15-20) to suppress microbial virulence and protect the integrity of the intestinal epithelium. The compound is nonmicrobiocidal and appears to be highly effective when tested both in vitro and in vivo. Structure functional analyses suggest that the hydrophobic bis-aromatic moiety at the polymer center is of particular importance to the biological function of Pi-PEG 15-20, beyond its phosphate content. Animal studies demonstrate that Pi-PEG prevents mortality in mice inoculated with multiple highly virulent pathogenic organisms from hospitalized patients in association with preservation of the core microbiome.
doi:10.1128/AAC.02183-13
PMCID: PMC3910877  PMID: 24277029
21.  Analysis of Serum Total and Free PSA Using Immunoaffinity Depletion Coupled to SRM: Correlation with Clinical Immunoassay Tests 
Journal of proteomics  2012;75(15):4747-4757.
Recently, selected reaction monitoring mass spectrometry (SRM-MS) has been more frequently applied to measure low abundance biomarker candidates in tissues and biofluids, owing to its high sensitivity and specificity, simplicity of assay configuration, and exceptional multiplexing capability. In this study, we report for the first time the development of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. Low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed good correlation in several independent clinical serum sample sets. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring candidate biomarkers in human blood, without the need to develop affinity reagents. Furthermore, the simultaneous measurement of multiple biomarkers, including the free and bound forms of PSA, can be performed in a single multiplexed analysis using high-resolution liquid chromatographic separation coupled with SRM-MS.
doi:10.1016/j.jprot.2012.01.035
PMCID: PMC3410652  PMID: 22846433
Selected reaction monitoring; immunoaffinity depletion; total PSA; free PSA; serum; immunoassay
22.  Cerebrospinal fluid proteome of patients with acute Lyme disease 
Journal of proteome research  2012;11(10):4814-4822.
During acute Lyme disease, bacteria can disseminate to the central nervous system (CNS) leading to the development of meningitis and other neurologic symptoms. Here we have analyzed pooled cerebrospinal fluid (CSF) allowing a deep view into the proteome for patients diagnosed with early-disseminated Lyme disease and CSF inflammation. Additionally, we analyzed individual patient samples and quantified differences in protein abundance employing label-free quantitative mass spectrometry based methods. We identified 108 proteins that differ significantly in abundance in patients with acute Lyme disease from controls. Comparison between infected patients and control subjects revealed differences in proteins in the CSF associated with cell death localized to brain synapses and others that likely originate from brain parenchyma.
doi:10.1021/pr300577p
PMCID: PMC3465517  PMID: 22900834
Proteomics; mass spectrometry; Lyme disease; cerebrospinal fluid; Lyme neuroborreliosis
23.  Gray Matter Is Targeted in First-Attack Multiple Sclerosis 
PLoS ONE  2013;8(9):e66117.
The cause of multiple sclerosis (MS), its driving pathogenesis at the earliest stages, and what factors allow the first clinical attack to manifest remain unknown. Some imaging studies suggest gray rather than white matter may be involved early, and some postulate this may be predictive of developing MS. Other imaging studies are in conflict. To determine if there was objective molecular evidence of gray matter involvement in early MS we used high-resolution mass spectrometry to identify proteins in the cerebrospinal fluid (CSF) of first-attack MS patients (two independent groups) compared to established relapsing remitting (RR) MS and controls. We found that the CSF proteins in first-attack patients were differentially enriched for gray matter components (axon, neuron, synapse). Myelin components did not distinguish these groups. The results support that gray matter dysfunction is involved early in MS, and also may be integral for the initial clinical presentation.
doi:10.1371/journal.pone.0066117
PMCID: PMC3769274  PMID: 24039694
24.  Reduced Insulin/Insulin-like Growth Factor-1 Signaling and Dietary Restriction Inhibit Translation but Preserve Muscle Mass in Caenorhabditis elegans *  
Molecular & Cellular Proteomics : MCP  2013;12(12):3624-3639.
Reduced signaling through the C. elegans insulin/insulin-like growth factor-1-like tyrosine kinase receptor daf-2 and dietary restriction via bacterial dilution are two well-characterized lifespan-extending interventions that operate in parallel or through (partially) independent mechanisms. Using accurate mass and time tag LC-MS/MS quantitative proteomics, we detected that the abundance of a large number of ribosomal subunits is decreased in response to dietary restriction, as well as in the daf-2(e1370) insulin/insulin-like growth factor-1-receptor mutant. In addition, general protein synthesis levels in these long-lived worms are repressed. Surprisingly, ribosomal transcript levels were not correlated to actual protein abundance, suggesting that post-transcriptional regulation determines ribosome content. Proteomics also revealed the increased presence of many structural muscle cell components in long-lived worms, which appeared to result from the prioritized preservation of muscle cell volume in nutrient-poor conditions or low insulin-like signaling. Activation of DAF-16, but not diet restriction, stimulates mRNA expression of muscle-related genes to prevent muscle atrophy. Important daf-2-specific proteome changes include overexpression of aerobic metabolism enzymes and general activation of stress-responsive and immune defense systems, whereas the increased abundance of many protein subunits of the proteasome core complex is a dietary-restriction-specific characteristic.
doi:10.1074/mcp.M113.027383
PMCID: PMC3861712  PMID: 24002365
25.  Proteome and Computational Analyses Reveal New Insights into the Mechanisms of Hepatitis C Virus Mediated Liver Disease Post-Transplantation 
Hepatology (Baltimore, Md.)  2012;56(1):28-38.
Liver transplant tissues offer the unique opportunity to model the longitudinal protein abundance changes occurring during hepatitis C virus (HCV)-associated liver disease progression in vivo. In this study, our goal was to identify molecular signatures, and potential key regulatory proteins, representative of the processes influencing early progression to fibrosis. We performed global protein profiling analyses on 24 liver biopsy specimens obtained from 15 HCV+ liver transplant recipients at 6 and/or 12 months post-transplantation. Differentially regulated proteins associated with early progression to fibrosis were identified by analysis of the area under the receiver operating characteristic curve (AUC). Analysis of serum metabolites was performed on samples obtained from an independent cohort of 60 HCV+ liver transplant patients. Computational modeling approaches were applied to identify potential key regulatory proteins of liver fibrogenesis. Among 4,324 proteins identified, 250 exhibited significant differential regulation in patients with rapidly progressive fibrosis. Patients with rapid fibrosis progression exhibited enrichment in differentially regulated proteins associated with various immune, hepatoprotective, and fibrogenic processes. The observed increase in pro-inflammatory activity and impairment in anti-oxidant defenses suggests that patients who develop significant liver injury experience elevated oxidative stresses. This was supported by an independent study demonstrating the altered abundance of oxidative stress associated serum metabolites in patients who develop severe liver injury. Computational modeling approaches further highlight a potentially important link between HCV-associated oxidative stress and epigenetic regulatory mechanisms impacting on liver fibrogenesis. In conclusion, our proteome and metabolome analyses provide new insights into the role for increased oxidative stress in the rapid fibrosis progression observed in HCV+ liver transplant recipients. These findings may prove useful in prognostic applications for predicting early progression to fibrosis.
doi:10.1002/hep.25649
PMCID: PMC3387320  PMID: 22331615
liver biopsy; systems biology; protein bottleneck

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