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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.  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
3.  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
4.  Glucocorticoid Receptor Hetero-Complex Gene STIP1 Is Associated with Improved Lung Function in Asthmatics Treated with Inhaled Corticosteroids 
Background
Corticosteroids exert their anti-inflammatory action by binding and activating the intracellular the glucocorticoid receptor (GR) hetero-complex.
Objective
Evaluate the genes HSPCB, HSPCA, STIP1, HSPA8, DNAJB1, PTGES3, FKBP5, and FKBP4 on corticosteroid response.
Methods
Caucasian asthmatics (382) randomized to once daily flunisolide or conventional inhaled corticosteroid therapy were genotyped. Outcome measures were baseline FEV1, % predicted FEV1, and % change in FEV1 after corticosteroid treatment. Multivariable analyses adjusted for age, gender, and height, were performed fitting the most appropriate genetic model based on quantitative mean derived from ANOVA models to determine if there was an independent effect of polymorphisms on change in FEV1 independent of baseline level.
Results
Positive recessive model correlations for STIP1 SNPs were observed for baseline FEV1 [rs4980524, p=0.009; rs6591838, p=0.0045; rs2236647, p=0.002; and rs2236648; p=0.013], baseline % predicted FEV1 [rs4980524, p=0.002; rs6591838, p=0.017; rs2236647, p=0.003; and rs2236648; p=0.008] ; % change in FEV1 at 4 weeks [rs4980524, p=0.044; rs6591838, p=0.016; rs2236647; p=0.01] and 8 weeks therapy [rs4980524, p=0.044; rs6591838, p=0.016; rs2236647; p=0.01]. Haplotypic associations were observed for baseline FEV1 and % change in FEV1 at 4 weeks therapy [p=0.05 and p=0.01, respectively]. Significant trends towards association were observed for baseline % predicted FEV1 and % change in FEV1 at 8 weeks therapy. Positive correlations between haplotypes and % change in FEV1 were also observed.
Conclusions
STIP1 genetic variations may play a role in regulating corticosteroid response in asthmatics with reduced lung function. Replication in a second asthma population is required to confirm these observations.
Clinical Implications
Identifying genes that regulate corticosteroid responses could allow a priori determination of individual responses to corticosteroid therapy, leading to more effective dosing and/or selection of drug therapies for treating asthma.
doi:10.1016/j.jaci.2009.01.049
PMCID: PMC4317788  PMID: 19254810
corticosteroid; pharmacogenetics; glucocorticoid receptor; SNP; heat shock protein; heat shock organizing protein; immunophilin
5.  Accounting for population variation in targeted proteomics 
Journal of proteome research  2013;13(1):321-323.
Individual proteomes typically differ from the reference human proteome at ~10,000 single amino acid variants. When viewed at the population scale, this individual variation results in a wide variety of protein sequences. In targeted proteomics experiments, such variability can confound accurate protein quantification. To assist researchers in identifying target peptides with high variability within the human population we have created the Population Variation plug-in for Skyline, which provides easy access to the polymorphisms stored in dbSNP. Given a set of peptides, the tool reports minor allele frequency for common polymorphisms. We highlight the importance of considering genetic variation by applying the tool to public datasets.
doi:10.1021/pr4011052
PMCID: PMC3923399  PMID: 24320210
MRM/SRM; genetic variation; bioinformatics; dbSNP
6.  Estimating personal exposures from ambient air-pollution measures: Using meta-analysis to assess measurement error 
Background
Although ambient concentrations of particulate matter ≤10μm (PM10) are often used as proxies for total personal exposure, correlation (r) between ambient and personal PM10 concentrations varies. Factors underlying this variation and its effect on health outcome-PM exposure relationships remain poorly understood.
Methods
We conducted a random-effects meta-analysis to estimate effects of study, participant and environmental factors on r; used the estimates to impute personal exposure from ambient PM10 concentrations among 4,012 non-smoking, diabetic participants in the Women’s Health Initiative clinical trial; and then estimated the associations of ambient and imputed personal PM10 concentrations with electrocardiographic measures such as heart rate variability.
Results
We identified fifteen studies (in years 1990-2009) of 342 participants in five countries. The median r was 0.46 (range = 0.13 to 0.72). There was little evidence of funnel-plot asymmetry but substantial heterogeneity of r, which increased 0.05 (95% confidence interval [CI]= 0.01 to 0.09) per 10 μg/m3 increase in mean ambient PM10 concentration. Substituting imputed personal exposure for ambient PM10 concentrations shifted mean percent changes in electrocardiographic measures per 10μg/m3 increase in exposure away from the null and decreased their precision, e.g. −2.0% (95% CI= −4.6% to 0.7%) versus −7.9% (−15.9% to 0.9%) for the standard deviation of normal-to-normal RR interval duration.
Conclusions
Analogous distributions and heterogeneity of r in extant meta-analyses of ambient and personal PM2.5 concentrations suggest that observed shifts in mean percent change and decreases in precision may be generalizable across particle size.
doi:10.1097/EDE.0000000000000006
PMCID: PMC3973436  PMID: 24220191
7.  Picoelectrospray ionization mass spectrometry using narrow-bore chemically etched emitters 
Electrospray ionization mass spectrometry (ESI-MS) at flow rates below ~10 nL/min has been only sporadically explored due to difficulty in reproducibly fabricating emitters that can operate at lower flow rates. Here we demonstrate narrow orifice chemically etched emitters for stable electrospray at flow rates as low as 400 pL/min. Depending on the analyte concentration, we observe two types of MS signal response as a function of flow rate. At low concentrations, an optimum flow rate is observed slightly above 1 nL/min, while the signal decreases monotonically with decreasing flow rates at higher concentrations. For example, consumption of 500 zmol of sample yielded signal-to-noise ratios ~10 for some peptides. In spite of lower MS signal, the ion utilization efficiency increases exponentially with decreasing flow rate in all cases. Significant variations in ionization efficiency were observed within this flow rate range for an equimolar mixture of peptides, indicating that ionization efficiency is an analyte-dependent characteristic for the present experimental conditions. Mass-limited samples benefit strongly from the use of low flow rates and avoiding unnecessary sample dilution. These findings have important implications for the analysis of trace biological samples.
doi:10.1007/s13361-013-0749-z
PMCID: PMC3945158  PMID: 24122304
8.  Transcriptomic and proteomic dynamics in the metabolism of a diazotrophic cyanobacterium, Cyanothece sp. PCC 7822 during a diurnal light–dark cycle 
BMC Genomics  2014;15(1):1185.
Background
Cyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light–dark cycle. Utilizing transcriptomic and proteomic methods, we quantified the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions.
Results
By combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four time points across a 24 hour light–dark cycle. Photosynthesis, nitrogen fixation, and carbon storage relevant genes were expressed during the preceding light or dark period, concurrent with measured nitrogenase activity in the late light period. We describe many instances of disparity in peak mRNA and protein abundances, and strong correlation of light dependent expression of both antisense and CRISPR-related gene expression. The proteins for nitrogenase and the pentose phosphate pathway were highest in the dark, whereas those for glycolysis and the TCA cycle were more prominent in the light. Interestingly, one copy of the psbA gene encoding the photosystem II (PSII) reaction center protein D1 (psbA4) was highly upregulated only in the dark. This protein likely cannot catalyze O2 evolution and so may be used by the cell to keep PSII intact during N2 fixation. The CRISPR elements were found exclusively at the ends of the large plasmid and we speculate that their presence is crucial to the maintenance of this plasmid.
Conclusions
This investigation of parallel transcriptional and translational activity within Cyanothece sp. PCC 7822 provided quantitative information on expression levels of metabolic pathways relevant to engineering efforts. The identification of expression patterns for both mRNA and protein affords a basis for improving biofuel production in this strain and for further genetic manipulations. Expression analysis of the genes encoded on the 6 plasmids provided insight into the possible acquisition and maintenance of some of these extra-chromosomal elements.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1185) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-1185
PMCID: PMC4320622  PMID: 25547186
Cyanothece; Cyanobacteria; RNA-Seq; N2 fixation; Proteomics; Butanol; CRISPR
9.  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
10.  The effect of external ankle support on the kinematics and kinetics of the lower limb during a side step cutting task in netballers 
Background
Excessive knee valgus moments are considered to be a risk factor for non-contact injuries in female athletes. Knee injuries are highly prevalent in netballers and are significant in terms of cost and disability. The aim of the study was to identify if changes in external ankle support mechanisms effect the range of motion and loading patterns at the ankle and knee joint during a sidestep cutting manoeuvre in high performance netball players.
Methods
Netballers with no previously diagnosed ankle or knee injury (n = 10) were recruited from NSW Institute of Sport netball programme. Kinematic and kinetic data were collected simultaneously using a 3-D Motion Analysis System and a force platform to measure ground reaction forces. Players performed repeated side step cutting manoeuvres whilst wearing a standard netball shoe, the same shoe with a lace-up brace and a high-top shoe.
Results
The brace condition significantly reduced ankle joint ROM in the sagittal plane by 8.9° ± 2.4 when compared to the standard netball shoe (p = 0.013). No other significant changes were seen between conditions for either kinematic or kinetic data. All shoe conditions did however produce knee valgus moments throughout the cutting cycle that were greater than those considered excessive in the previous literature (0.59 Nm/kg-Bwt).
Conclusions
The results show that an external ankle support brace can be used to reduce the ROM at the ankle in the sagittal plane without affecting the loading of the joints of the lower limb. Internal varus moments generated at the knee during the task were however greater than values reported in the literature to classify excessive knee joint moments, regardless of the condition. All netballers exhibited lower extremity patterns and alignments previously associated with increased peak external valgus moments including; increasing hip abduction, peak hip flexion and internal rotation during early contact and high laterally directed ground reaction forces. Increased external valgus knee loads have been strongly linked to the development of non-contact injuries at the knee in female athletes and could highlight a potential mechanism for the development non-contact knee injuries in netballers performing side step cutting tasks.
doi:10.1186/2052-1847-6-42
PMCID: PMC4297419  PMID: 25598984
Netball; Sidestep cutting; Biomechanics; External ankle support; Knee joint loading; Internal valgus moment
11.  Proteogenomic strategies for identification of aberrant cancer peptides using large-scale Next Generation Sequencing data 
Proteomics  2014;14(0):2719-2730.
Cancer is driven by the acquisition of somatic DNA lesions. Distinguishing the early driver mutations from subsequent passenger mutations is key to molecular sub-typing of cancers, understanding cancer progression, and the discovery of novel biomarkers. The advances of genomics technologies (whole-genome exome, and transcript sequencing, collectively referred to as NGS(Next Gengeration Sequencing)) have fueled recent studies on somatic mutation discovery. However, the vision is challenged by the complexity, redundancy, and errors in genomic data, and the difficulty of investigating the proteome translated portion of aberrant genes using only genomic approaches. Combination of proteomic and genomic technologies are increasingly being employed.
Various strategies have been employed to allow the usage of large scale NGS data for conventional MS/MS searches. This paper provides a discussion of applying different strategies relating to large database search, and FDR(False Discovery Rate) based error control, and their implication to cancer proteogenomics. Moreover, it extends and develops the idea of a unified genomic variant database that can be searched by any mass spectrometry sample. A total of 879 BAM files downloaded from TCGA repository were used to create a 4.34 GB unified FASTA database which contained 2, 787, 062 novel splice junctions, 38, 464 deletions, 1, 105 insertions, and 182, 302 substitutions. Proteomic data from a single ovarian carcinoma sample (439, 858 spectra) was searched against the database. By applying the most conservative FDR measure, we have identified 524 novel peptides and 65, 578 known peptides at 1% FDR threshold. The novel peptides include interesting examples of doubly mutated peptides, frame-shifts, and non-sample-recruited mutations, which emphasize the strength of our approach.
doi:10.1002/pmic.201400206
PMCID: PMC4256132  PMID: 25263569
12.  The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation 
Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ)-based LC–MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium vs sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.
doi:10.1016/j.fgb.2014.05.005
PMCID: PMC4247816  PMID: 24881580
Neurospora crassa; Proteome; Phosphoproteome; Cellulase; Carbon starvation; Plant biomass
13.  Genome-wide copy number variation analysis of a Branchio-Oto-Renal syndrome cohort identifies a recombination hotspot and implicates new candidate genes 
Human genetics  2013;132(12):10.1007/s00439-013-1338-8.
Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial arch anomalies, hearing loss and renal dysmorphology. Although haploinsufficiency of EYA1 and SIX1 are known to cause BOR, copy number variation analysis has only been performed on a limited number of BOR patients. In this study, we used high-resolution array-based comparative genomic hybridization (aCGH) on 32 BOR probands negative for coding-sequence and splice-site mutations in known BOR-causing genes to identify potential disease-causing genomic rearrangements. Of the >1,000 rare and novel copy number variants (CNVs) we identified, four were heterozygous deletions of EYA1 and several downstream genes that had nearly identical breakpoints associated with retroviral sequence blocks, suggesting that non-allelic homologous recombination seeded by this recombination hotspot is important in the pathogenesis of BOR. A different heterozygous deletion removing the last exon of EYA1 was identified in an additional proband. Thus in total 5 probands (14%) had deletions of all or part of EYA1. Using a novel disease-gene prioritization strategy that includes network analysis of genes associated with other deletions suggests that SHARPIN (Sipl1), FGF3 and the HOXA gene cluster may contribute to the pathogenesis of BOR.
doi:10.1007/s00439-013-1338-8
PMCID: PMC3830662  PMID: 23851940
array CGH; EYA1; birth defects; Branchio-oto-renal syndrome; copy number variation
14.  Improving the Sensitivity of Mass Spectrometry by Using a New Sheath Flow Electrospray Emitter Array at Subambient Pressures 
Arrays of chemically etched emitters with individualized sheath gas capillaries were developed to enhance electrospray ionization (ESI) efficiency at subambient pressures. By incorporating the new emitter array in a subambient pressure ionization with nanoelectrospray (SPIN) source, both ionization efficiency and ion transmission efficiency were significantly increased, providing enhanced sensitivity in mass spectrometric analyses. The SPIN source eliminates the major ion losses of conventional ESI-mass spectrometry (MS) interfaces by placing the emitter in the first reduced pressure region of the instrument. The new ESI emitter array design developed in this study allows individualized sheath gas around each emitter in the array making it possible to generate an array of uniform and stable electrosprays in the subambient pressure (10 to 30 Torr) environment for the first time. The utility of the new emitter arrays was demonstrated by coupling the emitter array/SPIN source with a time of flight (TOF) mass spectrometer. The instrument sensitivity was compared under different ESI source and interface configurations including a standard atmospheric pressure single ESI emitter/heated capillary, single emitter/SPIN and multi-emitter/SPIN configurations using an equimolar solution of 9 peptides. The highest instrument sensitivity was observed using the multi-emitter/SPIN configuration in which the sensitivity increased with the number of emitters in the array. Over an order of magnitude MS sensitivity improvement was achieved using multi-emitter/SPIN as compared to using the standard atmospheric pressure single ESI emitter/heated capillary interface.
doi:10.1007/s13361-014-0856-5
PMCID: PMC4177967  PMID: 24676894
15.  Detecting and Removing Data Artifacts in Hadamard Transform Ion Mobility-Mass Spectrometry Measurements 
Applying Hadamard transform multiplexing to ion mobility separations (IMS) can significantly improve the signal-to-noise ratio and throughput for IMS coupled mass spectrometry (MS) measurements by increasing the ion utilization efficiency. However, it has been determined that fluctuations in ion intensity as well as spatial shifts in the multiplexed data lower the signal-to-noise ratios and appear as noise in downstream processing of the data. To address this problem, we have developed a novel algorithm that discovers and eliminates data artifacts. The algorithm employs an analytical approach to identify and remove artifacts from the data, decreasing the likelihood of false identifications in subsequent data processing. Following application of the algorithm, IMS-MS measurement sensitivity is greatly increased and artifacts that previously limited the utility of applying the Hadamard transform to IMS are avoided.
doi:10.1007/s13361-014-0895-y
PMCID: PMC4223016  PMID: 24796262
Ion mobility; Computational proteomics; Hadamard transform; Bioinformatics; Sensitivity; Data analysis
16.  Evoked EMG versus Muscle Torque during Fatiguing Functional Electrical Stimulation-Evoked Muscle Contractions and Short-Term Recovery in Individuals with Spinal Cord Injury 
Sensors (Basel, Switzerland)  2014;14(12):22907-22920.
This study investigated whether the relationship between muscle torque and m-waves remained constant after short recovery periods, between repeated intervals of isometric muscle contractions induced by functional electrical stimulation (FES). Eight subjects with spinal cord injury (SCI) were recruited for the study. All subjects had their quadriceps muscles group stimulated during three sessions of isometric contractions separated by 5 min of recovery. The evoked-electromyographic (eEMG) signals, as well as the produced torque, were synchronously acquired during the contractions and during short FES bursts applied during the recovery intervals. All analysed m-wave variables changed progressively throughout the three contractions, even though the same muscle torque was generated. The peak to peak amplitude (PtpA), and the m-wave area (Area) were significantly increased, while the time between the stimulus artefact and the positive peak (PosT) were substantially reduced when the muscles became fatigued. In addition, all m-wave variables recovered faster and to a greater extent than did torque after the recovery intervals. We concluded that rapid recovery intervals between FES-evoked exercise sessions can radically interfere in the use of m-waves as a proxy for torque estimation in individuals with SCI. This needs to be further investigated, in addition to seeking a better understanding of the mechanisms of muscle fatigue and recovery.
doi:10.3390/s141222907
PMCID: PMC4299045  PMID: 25479324
evoked EMG; fatigue; torque; FES contraction; spinal cord injured
17.  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
18.  Increasing Confidence of LC-MS Identifications by Utilizing Ion Mobility Spectrometry 
Ion mobility spectrometry in conjunction with liquid chromatography separations and mass spectrometry offers a range of new possibilities for analyzing complex biological samples. To fully utilize the information obtained from these three measurement dimensions, informatics tools based on the accurate mass and time tag methodology were modified to incorporate ion mobility spectrometry drift times for peptides observed in human serum. In this work a reference human serum database was created for 12,139 peptides and populated with the monoisotopic mass, liquid chromatography normalized elution time, and ion mobility spectrometry drift time(s) for each. We demonstrate that the use of three dimensions for peak matching during the peptide identification process resulted in an increased numbers of identifications and a lower false discovery rate relative to only using the mass and normalized elution time dimensions.
doi:10.1016/j.ijms.2013.06.028
PMCID: PMC4114398  PMID: 25089116
ion mobility spectrometry; mass spectrometry; peptide identification; LC-MS; LC-IMS-MS; AMT tag; false discovery rate; drift time
19.  LC-IMS-MS Feature Finder: detecting multidimensional liquid chromatography, ion mobility and mass spectrometry features in complex datasets 
Bioinformatics  2013;29(21):2804-2805.
Motivation: The addition of ion mobility spectrometry to liquid chromatography-mass spectrometry experiments requires new, or updated, software tools to facilitate data processing.
Results: We introduce a command line software application LC-IMS-MS Feature Finder that searches for molecular ion signatures in multidimensional liquid chromatography-ion mobility spectrometry-mass spectrometry (LC-IMS-MS) data by clustering deisotoped peaks with similar monoisotopic mass, charge state, LC elution time and ion mobility drift time values. The software application includes an algorithm for detecting and quantifying co-eluting chemical species, including species that exist in multiple conformations that may have been separated in the IMS dimension.
Availability: LC-IMS-MS Feature Finder is available as a command-line tool for download at http://omics.pnl.gov/software/LC-IMS-MS_Feature_Finder.php. The Microsoft.NET Framework 4.0 is required to run the software. All other dependencies are included with the software package. Usage of this software is limited to non-profit research to use (see README).
Contact: rds@pnnl.gov
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btt465
PMCID: PMC3799467  PMID: 24008421
20.  Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid 
Analytical and bioanalytical chemistry  2014;406(28):7117-7125.
Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 μL) due to low yields stemming from losses caused by nonspecific binding to the column matrix and concentration of large eluent volumes. Additionally, the cost of the depletion media can be prohibitive for larger-scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large-scale studies. We characterized the performance of a 346 μL column volume microscale depletion system, using four different flow rates to determine the most effective depletion conditions for ~6-μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10-mL depletion column served as the control. Results showed depletion efficiency of the microscale column increased as flow rate decreased, and that our microdepletion was reproducible. In an initial application, a 600-μL sample of human cerebrospinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.
doi:10.1007/s00216-014-8058-3
PMCID: PMC4208972  PMID: 25192788
Microscale depletion; IgY-14 immunoaffinity resin; Human plasma; Cerebrospinal fluid; MS
21.  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
22.  Experimental Evaluation and Optimization of Structures for Lossless Ion Manipulations for Ion Mobility Spectrometry with Time-of-Flight Mass Spectrometry 
Analytical chemistry  2014;86(18):9169-9176.
We report on the performance of structures for lossless ion manipulation (SLIM) as a means for transmitting ions and performing ion mobility separations (IMS). Ions were successfully transferred from an electrospray ionization (ESI) source to the TOF MS analyzer by means of a linear SLIM, demonstrating lossless ion transmission and an alternative arrangement including a 90° turn. First, the linear geometry was optimized for radial confinement by tuning RF on the central “rung” electrodes and potentials on the DC-only guard electrodes. Selecting an appropriate DC guard bias (2–6 V) and RF amplitude (≥160 Vp-p at 750 kHz) resulted in the greatest ion intensities. Close to ideal IMS resolving power was maintained over a significant range of applied voltages. Second, the 90° turn was optimized for radial confinement by tuning RF on the rung electrodes and DC on the guard electrodes. However, both resolving power and ion transmission showed a dependence on these voltages, and the best conditions for both were >300 Vp-p RF (685 kHz) and 7–11 V guard DC bias. Both geometries provide IMS resolving powers at the theoretical limit (R ~ 58), showing that degraded resolution from a “racetrack” effect from turning around a corner can be successfully avoided, and the capability also was maintained for essentially lossless ion transmission.
doi:10.1021/ac502055e
PMCID: PMC4165449  PMID: 25152066
23.  Protein and microRNA biomarkers from lavage, urine, and serum in military personnel evaluated for dyspnea 
BMC Medical Genomics  2014;7:58.
Background
We have identified candidate protein and microRNA (miRNA) biomarkers for dyspnea by studying serum, lavage fluid, and urine from military personnel who reported serious respiratory symptoms after they were deployed to Iraq or Afghanistan.
Methods
Forty-seven soldiers with the complaint of dyspnea who enrolled in the STudy of Active Duty Military Personnel for Environmental Dust Exposure (STAMPEDE) underwent comprehensive pulmonary evaluations at the San Antonio Military Medical Center. The evaluation included fiber-optic bronchoscopy with bronchoalveolar lavage. The clinical findings from the STAMPEDE subjects pointed to seven general underlying diagnoses or findings including airway hyperreactivity, asthma, low diffusivity of carbon monoxide, and abnormal cell counts. The largest category was undiagnosed. As an exploratory study, not a classification study, we profiled proteins or miRNAs in lavage fluid, serum, or urine in this group to look for any underlying molecular patterns that might lead to biomarkers. Proteins in lavage fluid and urine were identified by accurate mass tag (database-driven) proteomics methods while miRNAs were profiled by a hybridization assay applied to serum, urine, and lavage fluid.
Results
Over seventy differentially expressed proteins were reliably identified both from lavage and from urine in forty-eight dyspnea subjects compared to fifteen controls with no known lung disorder. Six of these proteins were detected both in urine and lavage. One group of subjects was distinguished from controls by expressing a characteristic group of proteins. A related group of dyspnea subjects expressed a unique group of miRNAs that included one miRNA that was differentially overexpressed in all three fluids studied. The levels of several miRNAs also showed modest but direct associations with several standard clinical measures of lung health such as forced vital capacity or gas exchange efficiency.
Conclusions
Candidate proteins and miRNAs associated with the general diagnosis of dyspnea have been identified in subjects with differing medical diagnoses. Since these markers can be measured in readily obtained clinical samples, further studies are possible that test the value of these findings in more formal classification or case–control studies in much larger cohorts of subjects with specific lung diseases such as asthma, emphysema, or some other well-defined lung disease.
doi:10.1186/1755-8794-7-58
PMCID: PMC4193960  PMID: 25282157
Dyspnea; Biomarkers; Lavage fluid; Serum; Urine; Proteomics; MicroRNAs
24.  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
25.  Connecting Genomic Alterations to Cancer Biology with Proteomics: The NCI Clinical Proteomic Tumor Analysis Consortium 
Cancer discovery  2013;3(10):1108-1112.
The National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) is applying latest generation of proteomic technologies to genomically annotated tumors from The Cancer Genome Atlas (TCGA) program, a joint initiative of the NCI and National Human Genome Research Institute. By providing a fully integrated accounting of DNA, RNA and protein abnormalities in individual tumors, these datasets will illuminate the complex relationship between genomic abnormalities and cancer phenotypes, thus producing biological insights as well as a wave of novel candidate biomarkers and therapeutic targets amenable to verification using targeted mass spectrometry methods.
doi:10.1158/2159-8290.CD-13-0219
PMCID: PMC3800055  PMID: 24124232
Gene Expression; Cancer Proteomics; Protein Phosphorylation; Mass Spectrometry; Cancer Genome Atlas

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