Dean Billheimer

Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography−Tandem Mass Spectrometry

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.

Prognostic and Pathogenetic Value of Combining Clinical and Biochemical Indices in Patients With Acute Lung Injury

BACKGROUND No single clinical or biologic marker reliably predicts clinical outcomes in acute lung injury (ALI)/ARDS. We hypothesized that a combination of biologic and clinical markers would be superior to either biomarkers or clinical factors alone in predicting ALI/ARDS mortality and would provide insight into the pathogenesis of clinical ALI/ARDS. METHODS Eight biologic markers that reflect endothelial and epithelial injury, inflammation, and coagulation (von Willebrand factor antigen, surfactant protein D [SP-D]), tumor necrosis factor receptor-1, interleukin [IL]-6, IL-8, intercellular adhesion molecule-1, protein C, plasminogen activator inhibitor-1) were measured in baseline plasma from 549 patients in the ARDSNet trial of low vs high positive end-expiratory pressure. Mortality was modeled with multivariable logistic regression. Predictors were selected using backward elimination. Comparisons between candidate models were based on the receiver operating characteristics (ROC) and tests of integrated discrimination improvement. RESULTS Clinical predictors (Acute Physiology And Chronic Health Evaluation III [APACHE III], organ failures, age, underlying cause, alveolar-arterial oxygen gradient, plateau pressure) predicted mortality with an area under the ROC curve (AUC) of 0.82; a combination of eight biomarkers and the clinical predictors had an AUC of 0.85. The best performing biomarkers were the neutrophil chemotactic factor, IL-8, and SP-D, a product of alveolar type 2 cells, supporting the concept that acute inflammation and alveolar epithelial injury are important pathogenetic pathways in human ALI/ARDS. CONCLUSIONS A combination of biomarkers and clinical predictors is superior to clinical predictors or biomarkers alone for predicting mortality in ALI/ARDS and may be useful for stratifying patients in clinical trials. From a pathogenesis perspective, the degree of acute inflammation and alveolar epithelial injury are highly associated with the outcome of human ALI/ARDS.

Statistical Interpretation of Species Composition

The relative abundance of different species characterizes the structure of a biological community. We analyze an experiment addressing the relationship between omnivorous feeding linkages and community stability. Our goal is to determine whether communities with different predator compositions respond similarly to environmental disturbance. To evaluate these data, we develop a hierarchical statistical model that combines Aitchisons logistic normal distribution with a conditional multinomial observation distribution. In addition, we present an algebra for compositions that includes addition, scalar multiplication, and a metric for differences in compositions. The algebra aids interpretation of treatment effects, treatment interactions, and covariates. Markov chain Monte Carlo (MCMC) is used for inference in a Bayesian framework. Our experimental results indicate that a high degree of omnivory can help to stabilize community dynamics and prevent radical shifts in community composition. This result is at odds with classical food-web predictions, but agrees with recent theoretical formulations.

The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-β signaling

Inappropriate activation of developmental pathways is a well-recognized tumor-promoting mechanism. Here we show that overexpression of the homeoprotein Six1, normally a developmentally restricted transcriptional regulator, increases TGF-beta signaling in human breast cancer cells and induces an epithelial-mesenchymal transition (EMT) that is in part dependent on its ability to increase TGF-beta signaling. TGF-beta signaling and EMT have been implicated in metastatic dissemination of carcinoma. Accordingly, we used spontaneous and experimental metastasis mouse models to demonstrate that Six1 overexpression promotes breast cancer metastasis. In addition, we show that, like its induction of EMT, Six1-induced experimental metastasis is dependent on its ability to activate TGF-beta signaling. Importantly, in human breast cancers Six1 correlated with nuclear Smad3 and thus increased TGF-beta signaling. Further, breast cancer patients whose tumors overexpressed Six1 had a shortened time to relapse and metastasis and an overall decrease in survival. Finally, we show that the effects of Six1 on tumor progression likely extend beyond breast cancer, since its overexpression correlated with adverse outcomes in numerous other cancers including brain, cervical, prostate, colon, kidney, and liver. Our findings indicate that Six1, acting through TGF-beta signaling and EMT, is a powerful and global promoter of cancer metastasis.

In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy

Nonmelanoma skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are the most common skin cancers, presenting nearly as many cases as all other cancers combined. The current gold‐standard for clinical diagnosis of these lesions is histopathologic examination, an invasive, time‐consuming procedure. There is thus considerable interest in developing a real‐time, automated, noninvasive tool for nonmelanoma skin cancer diagnosis. In this study, we explored the capability of Raman microspectroscopy to provide differential diagnosis of BCC, SCC, inflamed scar tissue, and normal tissue in vivo.

Performance Metrics for Liquid Chromatography-Tandem Mass Spectrometry Systems in Proteomics Analyses

A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.

Interlaboratory Study Characterizing a Yeast Performance Standard for Benchmarking LC-MS Platform Performance

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize preanalytical and analytical variation in comparative proteomics experiments.

Global Analysis of Protein Damage by the Lipid Electrophile 4-Hydroxy-2-nonenal

Lipid peroxidation yields a variety of electrophiles, which are thought to contribute to the molecular pathogenesis of diseases involving oxidative stress, yet little is known of the scope of protein damage caused by lipid electrophiles. We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 μm HNE. HNE Michael adducts were biotinylated by reaction with biotinamidohexanoic acid hydrazide, captured with streptavidin, and the captured proteins were resolved by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and identified by liquid chromatography-tandem mass spectrometry. Of the 1500+ proteins identified, 417 displayed a statistically significant increase in adduction with increasing HNE exposure concentration. We further identified 18 biotin hydrazide-modified, HNE-adducted peptides by specific capture using anti-biotin antibody and analysis by high resolution liquid chromatography-tandem mass spectrometry. A subset of the identified HNE targets were validated with a streptavidin capture and immunoblotting approach, which enabled detection of adducts at HNE exposures as low as 1 μm. Protein interaction network analysis indicated several subsystems impacted by endogenous electrophiles in oxidative stress, including the 26 S proteasomal and chaperonin containing TCP-1 (CCT) systems involved in protein-folding and degradation, as well as the COP9 signalosome, translation initiation complex, and a large network of ribonucleoproteins. Global analyses of protein lipid electrophile adducts provide a systems-level perspective on the mechanisms of diseases involving oxidative stress.

Sestamibi Scanning and Minimally Invasive Radioguided Parathyroidectomy Without Intraoperative Parathyroid Hormone Measurement

ObjectiveTo evaluate the results of a large series of patients undergoing minimally invasive radioguided parathyroidectomy (MIRP) in which routine use of the intraoperative parathyroid hormone assay was not used, and to investigate characteristics between patients who had positive preoperative parathyroid scans versus those with negative scans. Summary Background DataThe technique of parathyroidectomy has traditionally involved bilateral exploration of the neck under general endotracheal anesthesia. Parathyroid imaging using technetium-99m sestamibi (MIBI) has evolved and can localize the adenomas in 80% to 90% of patients. The MIRP technique combines parathyroid scintigraphy with a hand-held gamma detector used intraoperatively to guide the surgeon to the adenoma in patients with positive MIBI scans. Central to this technique or other unilateral approaches is a positive MIBI scan. MethodsOne hundred seventy-three atients with primary hyperparathyroidism operated on by a single surgeon between January 1998 and July 2002 were included. One hundred twelve patients underwent the MIRP procedure and by definition had a positive preoperative parathyroid scan. The technique involved injecting 20 mCi MIBI 1 hour before the surgical procedure in patients who preoperatively had positive MIBI imaging. Patients had the choice of general or MAC anesthesia. Using an incision of less than 4 cm, the dissection to the adenoma was guided by the Navigator 11-mm probe. These 112 patients and 4 additional patients who for various reasons did not have the MIRP procedure yet had positive MIBI scans were compared to 57 patients who had clearly negative MIBI parathyroid imaging. ResultsFollow-up data were available for 108 of 112 patients who underwent MIRP. No patients had persistent hypercalcemia. The long-term success rate for the MIRP group was 98%. Fifty-two percent of the MIRP procedures were performed using MAC anesthesia. Overall, gland weight and serum PTH were related to the probability of a positive MIBI scan. Multiple logistic regression revealed that females were more likely to exhibit positive scans than were males for any fixed serum PTH level. For females, there was a significant relationship between increasing serum parathyroid hormone and a positive MIBI scan. Conversely, in males, the relationship between scan positivity and serum parathyroid hormone was weaker. ConclusionsThe MIRP technique without routine intraoperative serum parathyroid hormone measurement resulted in an excellent cure rate for primary hyperparathyroidism. As the MIRP technique as well as other techniques for unilateral cervical exploration are predicated on a positive parathyroid scan, the possible effect of gender on the sensitivity of MIBI scintigraphy for the detection of parathyroid adenomas warrants further investigation.

Analysis of Global and Absorption, Distribution, Metabolism, and Elimination Gene Expression in the Progressive Stages of Human Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is characterized by a series of pathological changes that range from simple fatty liver to nonalcoholic steatohepatitis (NASH). The objective of this study is to describe changes in global gene expression associated with the progression of human NAFLD. This study is focused on the expression levels of genes responsible for the absorption, distribution, metabolism, and elimination (ADME) of drugs. Differential gene expression between three clinically defined pathological groups—normal, steatosis, and NASH—was analyzed. Genome-wide mRNA levels in samples of human liver tissue were assayed with Affymetrix GeneChip Human 1.0ST arrays. A total of 11,633 genes exhibited altered expression out of 33,252 genes at a 5% false discovery rate. Most gene expression changes occurred in the progression from steatosis to NASH. Principal component analysis revealed that hepatic disease status was the major determinant of differential ADME gene expression rather than age or sex of sample donors. Among the 515 drug transporters and 258 drug-metabolizing enzymes (DMEs) examined, uptake transporters but not efflux transporters or DMEs were significantly over-represented in the number of genes down-regulated. These results suggest that uptake transporter genes are coordinately targeted for down-regulation at the global level during the pathological development of NASH and that these patients may have decreased drug uptake capacity. This coordinated regulation of uptake transporter genes is indicative of a hepatoprotective mechanism acting to prevent accumulation of toxic intermediates in disease-compromised hepatocytes.