Validating the ratio of insulin like growth factor binding protein 4 to sex hormone binding globulin as a prognostic predictor of preterm birth in Viet Nam: a case-cohort study.

OBJECTIVE: To validate a serum biomarker developed in the USA for preterm birth (PTB) risk stratification in Viet Nam. METHODS: Women with singleton pregnancies (n = 5000) were recruited between 19+0-23+6 weeks' gestation at Tu Du Hospital, Ho Chi Minh City. Maternal serum was collected from 19+0-22+6 weeks' gestation and participants followed to neonatal discharge. Relative insulin-like growth factor binding protein 4 (IGFBP4) and sex hormone binding globulin (SHBG) abundances were measured by mass spectrometry and their ratio compared between PTB cases and term controls. Discrimination (area under the receiver operating characteristic curve, AUC) and calibration for PTB <37 and <34 weeks' gestation were tested, with model tuning using clinical factors. Measured outcomes included all PTBs (any birth ≤37 weeks' gestation) and spontaneous PTBs (birth ≤37 weeks' gestation with clinical signs of initiation of parturition). RESULTS: Complete data were available for 4984 (99.7%) individuals. The cohort PTB rate was 6.7% (n = 335). We observed an inverse association between the IGFBP4/SHBG ratio and gestational age at birth (p = 0.017; AUC 0.60 [95% CI, 0.53-0.68]). Including previous PTB (for multiparous women) or prior miscarriage (for primiparous women) improved performance (AUC 0.65 and 0.70, respectively, for PTB <37 and <34 weeks' gestation). Optimal performance (AUC 0.74) was seen within 19-20 weeks' gestation, for BMI >21 kg/m2 and age 20-35 years. CONCLUSION: We have validated a novel serum biomarker for PTB risk stratification in a very different setting to the original study. Further research is required to determine appropriate ratio thresholds based on the prevalence of risk factors and the availability of resources and preventative therapies.

Clinical and economic evaluation of a proteomic biomarker preterm birth risk predictor: cost-effectiveness modeling of prenatal interventions applied to predicted higher-risk pregnancies within a large and diverse cohort.

OBJECTIVES: Preterm birth occurs in more than 10% of U.S. births and is the leading cause of U.S. neonatal deaths, with estimated annual costs exceeding $25 billion USD. Using real-world data, we modeled the potential clinical and economic utility of a prematurity-reduction program comprising screening in a racially and ethnically diverse population with a validated proteomic biomarker risk predictor, followed by case management with or without pharmacological treatment. METHODS: The ACCORDANT microsimulation model used individual patient data from a prespecified, randomly selected sub-cohort (N = 847) of a multicenter, observational study of U.S. subjects receiving standard obstetric care with masked risk predictor assessment (TREETOP; NCT02787213). All subjects were included in three arms across 500 simulated trials: standard of care (SoC, control); risk predictor/case management comprising increased outreach, education and specialist care (RP-CM, active); and multimodal management (risk predictor/case management with pharmacological treatment) (RP-MM, active). In the active arms, only subjects stratified as higher risk by the predictor were modeled as receiving the intervention, whereas lower-risk subjects received standard care. Higher-risk subjects' gestational ages at birth were shifted based on published efficacies, and dependent outcomes, calibrated using national datasets, were changed accordingly. Subjects otherwise retained their original TREETOP outcomes. Arms were compared using survival analysis for neonatal and maternal hospital length of stay, bootstrap intervals for neonatal cost, and Fisher's exact test for neonatal morbidity/mortality (significance, p < .05). RESULTS: The model predicted improvements for all outcomes. RP-CM decreased neonatal and maternal hospital stay by 19% (p = .029) and 8.5% (p = .001), respectively; neonatal costs' point estimate by 16% (p = .098); and moderate-to-severe neonatal morbidity/mortality by 29% (p = .025). RP-MM strengthened observed reductions and significance. Point estimates of benefit did not differ by race/ethnicity. CONCLUSIONS: Modeled evaluation of a biomarker-based test-and-treat strategy in a diverse population predicts clinically and economically meaningful improvements in neonatal and maternal outcomes.

Preterm birth, defined as delivery before 37 weeks' gestation, is the leading cause of illness and death in newborns. In the United States, more than 10% of infants are born prematurely, and this rate is substantially higher in lower-income, inner-city and Black populations. Prematurity associates with greatly increased risk of short- and long-term medical complications and can generate significant costs throughout the lives of affected children. Annual U.S. health care costs to manage short- and long-term prematurity complications are estimated to exceed $25 billion.Clinical interventions, including case management (increased patient outreach, education and specialist care), pharmacological treatment and their combination can provide benefit to pregnancies at higher risk for preterm birth. Early and sensitive risk detection, however, remains a challenge.We have developed and validated a proteomic biomarker risk predictor for early identification of pregnancies at increased risk of preterm birth. The ACCORDANT study modeled treatments with real-world patient data from a racially and ethnically diverse U.S. population to compare the benefits of risk predictor testing plus clinical intervention for higher-risk pregnancies versus no testing and standard care. Measured outcomes included neonatal and maternal length of hospital stay, associated costs and neonatal morbidity and mortality. The model projected improved outcomes and reduced costs across all subjects, including ethnic and racial minority populations, when predicted higher-risk pregnancies were treated using case management with or without pharmacological treatment. The biomarker risk predictor shows high potential to be a clinically important component of risk stratification for pregnant women, leading to tangible gains in reducing the impact of preterm birth.

Better Estimation of Spontaneous Preterm Birth Prediction Performance through Improved Gestational Age Dating.

The clinical management of pregnancy and spontaneous preterm birth (sPTB) relies on estimates of gestational age (GA). Our objective was to evaluate the effect of GA dating uncertainty on the observed performance of a validated proteomic biomarker risk predictor, and then to test the generalizability of that effect in a broader range of GA at blood draw. In a secondary analysis of a prospective clinical trial (PAPR; NCT01371019), we compared two GA dating categories: both ultrasound and dating by last menstrual period (LMP) (all subjects) and excluding dating by LMP (excluding LMP). The risk predictor's performance was observed at the validated risk predictor threshold both in weeks 191/7-206/7 and extended to weeks 180/7-206/7. Strict blinding and independent statistical analyses were employed. The validated biomarker risk predictor showed greater observed sensitivity of 88% at 75% specificity (increases of 17% and 1%) in more reliably dated (excluding-LMP) subjects, relative to all subjects. Excluding dating by LMP significantly improved the sensitivity in weeks 191/7-206/7. In the broader blood draw window, the previously validated risk predictor threshold significantly stratified higher and lower risk of sPTB, and the risk predictor again showed significantly greater observed sensitivity in excluding-LMP subjects. These findings have implications for testing the performance of models aimed at predicting PTB.

Performance of a validated spontaneous preterm delivery predictor in South Asian and Sub-Saharan African women: a nested case control study.

OBJECTIVES: To address the disproportionate burden of preterm birth (PTB) in low- and middle-income countries, this study aimed to (1) verify the performance of the United States-validated spontaneous PTB (sPTB) predictor, comprised of the IBP4/SHBG protein ratio, in subjects from Bangladesh, Pakistan and Tanzania enrolled in the Alliance for Maternal and Newborn Health Improvement (AMANHI) biorepository study, and (2) discover biomarkers that improve performance of IBP4/SHBG in the AMANHI cohort. STUDY DESIGN: The performance of the IBP4/SHBG biomarker was first evaluated in a nested case control validation study, then utilized in a follow-on discovery study performed on the same samples. Levels of serum proteins were measured by targeted mass spectrometry. Differences between the AMANHI and U.S. cohorts were adjusted using body mass index (BMI) and gestational age (GA) at blood draw as covariates. Prediction of sPTB < 37 weeks and < 34 weeks was assessed by area under the receiver operator curve (AUC). In the discovery phase, an artificial intelligence method selected additional protein biomarkers complementary to IBP4/SHBG in the AMANHI cohort. RESULTS: The IBP4/SHBG biomarker significantly predicted sPTB < 37 weeks (n = 88 vs. 171 terms ≥ 37 weeks) after adjusting for BMI and GA at blood draw (AUC= 0.64, 95% CI: 0.57-0.71, p < .001). Performance was similar for sPTB < 34 weeks (n = 17 vs. 184 ≥ 34 weeks): AUC = 0.66, 95% CI: 0.51-0.82, p = .012. The discovery phase of the study showed that the addition of endoglin, prolactin, and tetranectin to the above model resulted in the prediction of sPTB < 37 with an AUC= 0.72 (95% CI: 0.66-0.79, p-value < .001) and prediction of sPTB < 34 with an AUC of 0.78 (95% CI: 0.67-0.90, p < .001). CONCLUSION: A protein biomarker pair developed in the U.S. may have broader application in diverse non-U.S. populations.

Clinical Validation of a Proteomic Biomarker Threshold for Increased Risk of Spontaneous Preterm Birth and Associated Clinical Outcomes: A Replication Study.

Preterm births are the leading cause of neonatal death in the United States. Previously, a spontaneous preterm birth (sPTB) predictor based on the ratio of two proteins, IBP4/SHBG, was validated as a predictor of sPTB in the Proteomic Assessment of Preterm Risk (PAPR) study. In particular, a proteomic biomarker threshold of -1.37, corresponding to a ~two-fold increase or ~15% risk of sPTB, significantly stratified earlier deliveries. Guidelines for molecular tests advise replication in a second independent study. Here we tested whether the significant association between proteomic biomarker scores above the threshold and sPTB, and associated adverse outcomes, was replicated in a second independent study, the Multicenter Assessment of a Spontaneous Preterm Birth Risk Predictor (TREETOP). The threshold significantly stratified subjects in PAPR and TREETOP for sPTB (p = 0.041, p = 0.041, respectively). Application of the threshold in a Kaplan-Meier analysis demonstrated significant stratification in each study, respectively, for gestational age at birth (p < 001, p = 0.0016) and rate of hospital discharge for both neonate (p < 0.001, p = 0.005) and mother (p < 0.001, p < 0.001). Above the threshold, severe neonatal morbidity/mortality and mortality alone were 2.2 (p = 0.0083,) and 7.4-fold higher (p = 0.018), respectively, in both studies combined. Thus, higher predictor scores were associated with multiple adverse pregnancy outcomes.

Performance of a proteomic preterm delivery predictor in a large independent prospective cohort.

BACKGROUND: Preterm birth remains a common and devastating complication of pregnancy. There remains a need for effective and accurate screening methods for preterm birth. Using a proteomic approach, we previously discovered and validated (Proteomic Assessment of Preterm Risk study, NCT01371019) a preterm birth predictor comprising a ratio of insulin-like growth factor-binding protein 4 to sex hormone-binding globulin. OBJECTIVE: To determine the performance of the ratio of insulin-like growth factor-binding protein 4 to sex hormone-binding globulin to predict both spontaneous and medically indicated very preterm births, in an independent cohort distinct from the one in which it was developed. STUDY DESIGN: This was a prospective observational study (Multicenter Assessment of a Spontaneous Preterm Birth Risk Predictor, NCT02787213) at 18 sites in the United States. Women had blood drawn at 170/7 to 216/7 weeks' gestation. For confirmation, we planned to analyze a randomly selected subgroup of women having blood drawn between 191/7 and 206/7 weeks' gestation, with the results of the remaining study participants blinded for future validation studies. Serum from participants was analyzed by mass spectrometry. Neonatal morbidity and mortality were analyzed using a composite score by a method from the PREGNANT trial (NCT00615550, Hassan et al). Scores of 0-3 reflect increasing numbers of morbidities or length of neonatal intensive care unit stay, and 4 represents perinatal mortality. RESULTS: A total of 5011 women were enrolled, with 847 included in this planned substudy analysis. There were 9 preterm birth cases at <320/7 weeks' gestation and 838 noncases at ≥320/7 weeks' gestation; 21 of 847 infants had neonatal composite morbidity and mortality index scores of ≥3, and 4 of 21 had a score of 4. The ratio of insulin-like growth factor-binding protein 4 to sex hormone-binding globulin ratio was substantially higher in both preterm births at <320/7 weeks' gestation and there were more severe neonatal outcomes. The ratio of insulin-like growth factor-binding protein 4 to sex hormone-binding globulin ratio was significantly predictive of birth at <320/7 weeks' gestation (area under the receiver operating characteristic curve, 0.71; 95% confidence interval, 0.55-0.87; P=.016). Stratification by body mass index, optimized in the previous validation study (22

Development and validation of a spontaneous preterm delivery predictor in asymptomatic women.

BACKGROUND: Preterm delivery remains the leading cause of perinatal mortality. Risk factors and biomarkers have traditionally failed to identify the majority of preterm deliveries. OBJECTIVE: To develop and validate a mass spectrometry-based serum test to predict spontaneous preterm delivery in asymptomatic pregnant women. STUDY DESIGN: A total of 5501 pregnant women were enrolled between 17(0/7) and 28(6/7) weeks gestational age in the prospective Proteomic Assessment of Preterm Risk study at 11 sites in the United States between 2011 and 2013. Maternal blood was collected at enrollment and outcomes collected following delivery. Maternal serum was processed by a proteomic workflow, and proteins were quantified by multiple reaction monitoring mass spectrometry. The discovery and verification process identified 2 serum proteins, insulin-like growth factor-binding protein 4 (IBP4) and sex hormone-binding globulin (SHBG), as predictors of spontaneous preterm delivery. We evaluated a predictor using the log ratio of the measures of IBP4 and SHBG (IBP4/SHBG) in a clinical validation study to classify spontaneous preterm delivery cases (<37(0/7) weeks gestational age) in a nested case-control cohort different from subjects used in discovery and verification. Strict blinding and independent statistical analyses were employed. RESULTS: The predictor had an area under the receiver operating characteristic curve value of 0.75 and sensitivity and specificity of 0.75 and 0.74, respectively. The IBP4/SHBG predictor at this sensitivity and specificity had an odds ratio of 5.04 for spontaneous preterm delivery. Accuracy of the IBP4/SHBG predictor increased using earlier case-vs-control gestational age cutoffs (eg, <35(0/7) vs ≥35(0/7) weeks gestational age). Importantly, higher-risk subjects defined by the IBP4/SHBG predictor score generally gave birth earlier than lower-risk subjects. CONCLUSION: A serum-based molecular predictor identifies asymptomatic pregnant women at risk of spontaneous preterm delivery, which may provide utility in identifying women at risk at an early stage of pregnancy to allow for clinical intervention. This early detection would guide enhanced levels of care and accelerate development of clinical strategies to prevent preterm delivery.

The Rhesus macaque (Macaca mulatta) sperm proteome.

Mass spectrometry based proteomics has facilitated sperm composition studies in several mammalian species but no studies have been undertaken in non-human primate species. Here we report the analysis of the 1247 proteins that comprise the Rhesus macaque (Macaca mulatta) sperm proteome (termed the MacSP). Comparative analysis with previously characterized mouse and human sperm proteomes reveals substantial levels of orthology (47% and 40% respectively) and widespread overlap of functional categories based on Gene Ontology analyses. Approximately 10% of macaque sperm genes (113/1247) are significantly under-expressed in the testis as compared with other tissues, which may reflect proteins specifically acquired during epididymal maturation. Phylogenetic and genomic analyses of three MacSP ADAMs (A-Disintegrin and Metalloprotease proteins), ADAM18-, 20- and 21-like, provides empirical support for sperm genes functioning in non-human primate taxa which have been subsequently lost in the lineages leading to humans. The MacSP contains proteasome proteins of the 20S core subunit, the 19S proteasome activator complex and an alternate proteasome activator PA200, raising the possibility that proteasome activity is present in mature sperm. Robust empirical characterization of the Rhesus sperm proteome should greatly expand the possibility for targeted molecular studies of spermatogenesis and fertilization in a commonly used model species for human infertility.

MultiAlign: a multiple LC-MS analysis tool for targeted omics analysis.

BACKGROUND: MultiAlign is a free software tool that aligns multiple liquid chromatography-mass spectrometry datasets to one another by clustering mass and chromatographic elution features across datasets. Applicable to both label-free proteomics and metabolomics comparative analyses, the software can be operated in several modes. For example, clustered features can be matched to a reference database to identify analytes, used to generate abundance profiles, linked to tandem mass spectra based on parent precursor masses, and culled for targeted liquid chromatography-tandem mass spectrometric analysis. MultiAlign is also capable of tandem mass spectral clustering to describe proteome structure and find similarity in subsequent sample runs. RESULTS: MultiAlign was applied to two large proteomics datasets obtained from liquid chromatography-mass spectrometry analyses of environmental samples. Peptides in the datasets for a microbial community that had a known metagenome were identified by matching mass and elution time features to those in an established reference peptide database. Results compared favorably with those obtained using existing tools such as VIPER, but with the added benefit of being able to trace clusters of peptides across conditions to existing tandem mass spectra. MultiAlign was further applied to detect clusters across experimental samples derived from a reactor biomass community for which no metagenome was available. Several clusters were culled for further analysis to explore changes in the community structure. Lastly, MultiAlign was applied to liquid chromatography-mass spectrometry-based datasets obtained from a previously published study of wild type and mitochondrial fatty acid oxidation enzyme knockdown mutants of human hepatocarcinoma to demonstrate its utility for analyzing metabolomics datasets. CONCLUSION: MultiAlign is an efficient software package for finding similar analytes across multiple liquid chromatography-mass spectrometry feature maps, as demonstrated here for both proteomics and metabolomics experiments. The software is particularly useful for proteomic studies where little or no genomic context is known, such as with environmental proteomics.

DanteR: an extensible R-based tool for quantitative analysis of -omics data.

MOTIVATION: The size and complex nature of mass spectrometry-based proteomics datasets motivate development of specialized software for statistical data analysis and exploration. We present DanteR, a graphical R package that features extensive statistical and diagnostic functions for quantitative proteomics data analysis, including normalization, imputation, hypothesis testing, interactive visualization and peptide-to-protein rollup. More importantly, users can easily extend the existing functionality by including their own algorithms under the Add-On tab. AVAILABILITY: DanteR and its associated user guide are available for download free of charge at http://omics.pnl.gov/software/. We have an updated binary source for the DanteR package up on our website together with a vignettes document. For Windows, a single click automatically installs DanteR along with the R programming environment. For Linux and Mac OS X, users must install R and then follow instructions on the DanteR website for package installation. CONTACT: rds@pnnl.gov.

Differential effects of AKT1(p.E17K) expression on human mammary luminal epithelial and myoepithelial cells.

Recently, we identified a somatic mutation in AKT1, which results in a glutamic acid to lysine substitution (p.Glu17Lys or E17K). E17K mutations appear almost exclusively in breast cancers of luminal origin. Cellular models involving cell lines such as human mammary epithelial and MCF10 are model systems that upon transformation lead to rare forms of human breast cancer. Hence, we studied the effects of E17K using a clinically pertinent luminal cell line model while providing evidence to explain why E17K mutations do not occur in the mammary myoepithelium. Thus the purpose of our study was to perform a functional and differential proteomics study to assess the role of AKT1(E17K) in the development of breast cancer. We used a set of genetically matched nontumorigenic and tumorigenic mammary luminal and myoepithelial cells. We demonstrated that in myoepithelial cells, expression of E17K inhibited growth, migration, and protein synthesis compared with wild-type AKT1. In luminal cells, E17K enhanced cell survival and migration, possibly offering a selective advantage in this type of cell. However, antineoplastic effects of E17K in luminal cells, such as inhibition of growth and protein synthesis, may ultimately be associated with favorable prognosis. Our study illustrates the importance of cellular context in determining phenotypic effects of putative oncogenic mutations.

Diurnal rhythms result in significant changes in the cellular protein complement in the cyanobacterium Cyanothece 51142.

Cyanothece sp. ATCC 51142 is a diazotrophic cyanobacterium notable for its ability to perform oxygenic photosynthesis and dinitrogen fixation in the same single cell. Previous transcriptional analysis revealed that the existence of these incompatible cellular processes largely depends on tightly synchronized expression programs involving ∼30% of genes in the genome. To expand upon current knowledge, we have utilized sensitive proteomic approaches to examine the impact of diurnal rhythms on the protein complement in Cyanothece 51142. We found that 250 proteins accounting for ∼5% of the predicted ORFs from the Cyanothece 51142 genome and 20% of proteins detected under alternating light/dark conditions exhibited periodic oscillations in their abundances. Our results suggest that altered enzyme activities at different phases during the diurnal cycle can be attributed to changes in the abundance of related proteins and key compounds. The integration of global proteomics and transcriptomic data further revealed that post-transcriptional events are important for temporal regulation of processes such as photosynthesis in Cyanothece 51142. This analysis is the first comprehensive report on global quantitative proteomics in a unicellular diazotrophic cyanobacterium and uncovers novel findings about diurnal rhythms.

The Drosophila melanogaster sperm proteome-II (DmSP-II).

Advances in mass spectrometry technology, high-throughput proteomics and genome annotations have resulted in significant increases in our molecular understanding of sperm composition. Using improved separation and detection methods and an updated genome annotation, a re-analysis of the Drosophila melanogaster sperm proteome (DmSP) has resulted in the identification of 956 sperm proteins. Comparative analysis with our previous proteomic dataset revealed 766 new proteins and an updated sperm proteome containing a total of 1108 proteins, termed the DmSP-II. This expanded dataset includes additional proteins with predicted sperm functions and confirms previous findings concerning the genomic organization of sperm loci. Bioinformatic and protein network analyses demonstrated high quality and reproducibility of proteome coverage across three replicate mass spectrometry runs. The use of whole-cell proteomics in conjunction with characterized phenotypes, functional annotations and pathway information has advanced our systems level understanding of sperm proteome functional networks.

Omic data from evolved E. coli are consistent with computed optimal growth from genome-scale models.

After hundreds of generations of adaptive evolution at exponential growth, Escherichia coli grows as predicted using flux balance analysis (FBA) on genome-scale metabolic models (GEMs). However, it is not known whether the predicted pathway usage in FBA solutions is consistent with gene and protein expression in the wild-type and evolved strains. Here, we report that >98% of active reactions from FBA optimal growth solutions are supported by transcriptomic and proteomic data. Moreover, when E. coli adapts to growth rate selective pressure, the evolved strains upregulate genes within the optimal growth predictions, and downregulate genes outside of the optimal growth solutions. In addition, bottlenecks from dosage limitations of computationally predicted essential genes are overcome in the evolved strains. We also identify regulatory processes that may contribute to the development of the optimal growth phenotype in the evolved strains, such as the downregulation of known regulons and stringent response suppression. Thus, differential gene and protein expression from wild-type and adaptively evolved strains supports observed growth phenotype changes, and is consistent with GEM-computed optimal growth states.

Liquid Chromatography Mass Spectrometry-Based Proteomics: Biological and Technological Aspects.

Mass spectrometry-based proteomics has become the tool of choice for identifying and quantifying the proteome of an organism. Though recent years have seen a tremendous improvement in instrument performance and the computational tools used, significant challenges remain, and there are many opportunities for statisticians to make important contributions. In the most widely used "bottom-up" approach to proteomics, complex mixtures of proteins are first subjected to enzymatic cleavage, the resulting peptide products are separated based on chemical or physical properties and analyzed using a mass spectrometer. The two fundamental challenges in the analysis of bottom-up MS-based proteomics are: (1) Identifying the proteins that are present in a sample, and (2) Quantifying the abundance levels of the identified proteins. Both of these challenges require knowledge of the biological and technological context that gives rise to observed data, as well as the application of sound statistical principles for estimation and inference. We present an overview of bottom-up proteomics and outline the key statistical issues that arise in protein identification and quantification.

Novel Ser/Thr protein phosphatase 5 (PP5) regulated targets during DNA damage identified by proteomics analysis.

The DNA damage response likely includes a global phosphorylation signaling cascade process for sensing the damaged DNA condition and coordinating responses to cope with and repair the perturbed cellular state. We utilized a label-free liquid chromatography-mass spectrometry approach to evaluate changes in protein phosphorylation associated with PP5 activity during the DNA damage response. Biological replicate analyses of bleomycin-treated HeLa cells expressing either WT-PP5 or mutant inactive PP5 lead to the identification of six potential target proteins of PP5 action. Four of these putative targets have been previously reported to be involved in DNA damage responses. Using phospho-site specific antibodies, we confirmed that phosphorylation of one target, ribosomal protein S6, was selectively decreased in cells overexpressing catalytically inactive PP5. Our findings also suggest that PP5 may play a role in controlling translation and in regulating substrates for proline-directed kinases, such as MAP kinases and cyclin-dependent protein kinases that are involved in response to DNA damage.

DtaRefinery, a software tool for elimination of systematic errors from parent ion mass measurements in tandem mass spectra data sets.

Hybrid two-stage mass spectrometers capable of both highly accurate mass measurement and high throughput MS/MS fragmentation have become widely available in recent years, allowing for significantly better discrimination between true and false MS/MS peptide identifications by the application of a relatively narrow window for maximum allowable deviations of measured parent ion masses. To fully gain the advantage of highly accurate parent ion mass measurements, it is important to limit systematic mass measurement errors. Based on our previous studies of systematic biases in mass measurement errors, here, we have designed an algorithm and software tool that eliminates the systematic errors from the peptide ion masses in MS/MS data. We demonstrate that the elimination of the systematic mass measurement errors allows for the use of tighter criteria on the deviation of measured mass from theoretical monoisotopic peptide mass, resulting in a reduction of both false discovery and false negative rates of peptide identification. A software implementation of this algorithm called DtaRefinery reads a set of fragmentation spectra, searches for MS/MS peptide identifications using a FASTA file containing expected protein sequences, fits a regression model that can estimate systematic errors, and then corrects the parent ion mass entries by removing the estimated systematic error components. The output is a new file with fragmentation spectra with updated parent ion masses. The software is freely available.

Combined pulsed-Q dissociation and electron transfer dissociation for identification and quantification of iTRAQ-labeled phosphopeptides.

Here, we report a new approach that integrates pulsed Q dissociation (PQD) and electron transfer dissociation (ETD) techniques for confident and quantitative identification of iTRAQ-labeled phosphopeptides. The use of isobaric tags for relative and absolute quantification enables a high-throughput quantification of peptides via reporter ion signals in the low m/z range of tandem mass spectra. PQD, a form of ion trap collision activated dissociation, allows for detection of low mass-to-charge fragment ions, and electron transfer dissociation is especially useful for sequencing peptides that contain post-translational modifications. Analysis of the phosphoproteome of human fibroblast cells using a sensitive linear ion trap mass spectrometer demonstrated that this hybrid approach improves both identification and quantification of phosphopeptides. ETD improved phosphopeptide identification, while PQD provides improved quantification of iTRAQ-labeled phosphopeptides.

Using systems biology to simplify complex disease: immune cartography.

What if there was a rapid, inexpensive, and accurate blood diagnostic that could determine which patients were infected, identify the organism(s) responsible, and identify patients who were not responding to therapy? We hypothesized that systems analysis of the transcriptional activity of circulating immune effector cells could be used to identify conserved elements in the host response to systemic inflammation, and furthermore, to discriminate between sterile and infectious etiologies. We review herein a validated, systems biology approach demonstrating that 1) abdominal and pulmonary sepsis diagnoses can be made in mouse models using microarray (RNA) data from circulating blood, 2) blood microarray data can be used to differentiate between the host response to Gram-negative and Gram-positive pneumonia, 3) the endotoxin response of normal human volunteers can be mapped at the level of gene expression, and 4) a similar strategy can be used in the critically ill to follow septic patients and quantitatively determine immune recovery. These findings provide the foundation of immune cartography and demonstrate the potential of this approach for rapidly diagnosing sepsis and identifying pathogens. Further, our data suggest a new approach to determine how specific pathogens perturb the physiology of circulating leukocytes in a cell-specific manner. Large, prospective clinical trails are needed to validate the clinical utility of leukocyte RNA diagnostics (e.g., the riboleukogram).

Large-scale multiplexed quantitative discovery proteomics enabled by the use of an (18)O-labeled "universal" reference sample.

The quantitative comparison of protein abundances across a large number of biological or patient samples represents an important proteomics challenge that needs to be addressed for proteomics discovery applications. Herein, we describe a strategy that incorporates a stable isotope (18)O-labeled "universal" reference sample as a comprehensive set of internal standards for analyzing large sample sets quantitatively. As a pooled sample, the (18)O-labeled "universal" reference sample is spiked into each individually processed unlabeled biological sample and the peptide/protein abundances are quantified based on (16)O/(18)O isotopic peptide pair abundance ratios that compare each unlabeled sample to the identical reference sample. This approach also allows for the direct application of label-free quantitation across the sample set simultaneously along with the labeling-approach (i.e., dual-quantitation) since each biological sample is unlabeled except for the labeled reference sample that is used as internal standards. The effectiveness of this approach for large-scale quantitative proteomics is demonstrated by its application to a set of 18 plasma samples from severe burn patients. When immunoaffinity depletion and cysteinyl-peptide enrichment-based fractionation with high resolution LC-MS measurements were combined, a total of 312 plasma proteins were confidently identified and quantified with a minimum of two unique peptides per protein. The isotope labeling data was directly compared with the label-free (16)O-MS intensity data extracted from the same data sets. The results showed that the (18)O reference-based labeling approach had significantly better quantitative precision compared to the label-free approach. The relative abundance differences determined by the two approaches also displayed strong correlation, illustrating the complementary nature of the two quantitative methods. The simplicity of including the (18)O-reference for accurate quantitation makes this strategy especially attractive when a large number of biological samples are involved in a study where label-free quantitation may be problematic, for example, due to issues associated with instrument platform robustness. The approach will also be useful for more effectively discovering subtle abundance changes in broad systems biology studies.