Mu Wang

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low μg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.

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.

Sepsis: An integrated clinico-metabolomic model improves prediction of death in sepsis

A molecular signature, derived from integrated analysis of clinical data, the metabolome, and the proteome in prospective human studies, improved the prediction of death in patients with sepsis, potentially identifying a subset of patients who merit intensive treatment. Understanding Survival of the Fittest in Sepsis Differentiating mild infections from life-threatening ones is a complex decision that is made millions of times a year in U.S. emergency rooms. Should a patient be sent home with antibiotics and chicken soup? Or should he or she be hospitalized for intensive treatment? Sepsis—a serious infection that is associated with a generalized inflammatory response—is one of the leading causes of death. In two prospective clinical studies reported by Langley et al., patients arriving at four urban emergency departments with symptoms of sepsis were evaluated clinically and by analysis of their plasma proteome and metabolome. Survivors and nonsurvivors at 28 days were compared, and a molecular signature was detected that appeared to differentiate these outcomes—even as early as the time of hospital arrival. The signature was part of a large set of differences between these groups, showing that better energy-producing fatty acid catabolism was associated with survival of the fittest in sepsis. A test developed from the signature was able to predict sepsis survival and nonsurvival reproducibly and better than current methods. This test could help to make all important decisions in the emergency room more accurate. Sepsis is a common cause of death, but outcomes in individual patients are difficult to predict. Elucidating the molecular processes that differ between sepsis patients who survive and those who die may permit more appropriate treatments to be deployed. We examined the clinical features and the plasma metabolome and proteome of patients with and without community-acquired sepsis, upon their arrival at hospital emergency departments and 24 hours later. The metabolomes and proteomes of patients at hospital admittance who would ultimately die differed markedly from those of patients who would survive. The different profiles of proteins and metabolites clustered into the following groups: fatty acid transport and β-oxidation, gluconeogenesis, and the citric acid cycle. They differed consistently among several sets of patients, and diverged more as death approached. In contrast, the metabolomes and proteomes of surviving patients with mild sepsis did not differ from survivors with severe sepsis or septic shock. An algorithm derived from clinical features together with measurements of five metabolites predicted patient survival. This algorithm may help to guide the treatment of individual patients with sepsis.

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.

Multi-dimensional liquid chromatography in proteomics-A review

Proteomics is the large-scale study of proteins, particularly their expression, structures and functions. This still-emerging combination of technologies aims to describe and characterize all expressed proteins in a biological system. Because of upper limits on mass detection of mass spectrometers, proteins are usually digested into peptides and the peptides are then separated, identified and quantified from this complex enzymatic digest. The problem in digesting proteins first and then analyzing the peptide cleavage fragments by mass spectrometry is that huge numbers of peptides are generated that overwhelm direct mass spectral analyses. The objective in the liquid chromatography approach to proteomics is to fractionate peptide mixtures to enable and maximize identification and quantification of the component peptides by mass spectrometry. This review will focus on existing multidimensional liquid chromatographic (MDLC) platforms developed for proteomics and their application in combination with other techniques such as stable isotope labeling. We also provide some perspectives on likely future developments.

Label-free mass spectrometry-based protein quantification technologies in proteomic analysis

Major technological advances have made proteomics an extremely active field for biomarker discovery and validation in recent years. These improvements have lead to an increased emphasis on larger scale, faster and more efficient methods for protein biomarker discoveries in human tissues, cells and biofluids. However, most current proteomic methodologies for biomarker discovery and validation are not highly automated and generally labour intensive and expensive. Improved automation as well as software programs capable of handling a large amount of data are essential in order to reduce the cost of discovery and increase the throughput. In this review, we will discuss and describe the label-free mass spectrometry-based protein quantification technologies and a case study utilizing one of these methods for biomarker discovery.

Proteomic analysis of blastema formation in regenerating axolotl limbs

BackgroundFollowing amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa) through the mid-tibia/fibula of axolotl hind limbs.ResultsWe identified 309 unique proteins with significant fold change relative to controls (0 dpa), representing 10 biological process categories: (1) signaling, (2) Ca2+ binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) extracellular matrix (ECM), (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5), a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation.ConclusionOur data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR). Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures.

Serum proteomics and biomarker discovery across the spectrum of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD), ranging from relatively benign simple steatosis to progressive nonalcoholic steatohepatitis (NASH) and fibrosis, is an increasingly common chronic liver disease. Liver biopsy is currently the only reliable tool for staging the subtypes of NAFLD; therefore, noninvasive serum biomarkers for evaluation of liver disease and fibrosis are urgently needed. We performed this study to describe changes in the serum proteome and identify biomarker candidates in serum samples from 69 patients with varying stages of NAFLD (simple steatosis, NASH, and NASH with advanced bridging [F3/F4] fibrosis) and 16 obese controls. Using a label‐free mass spectrometry‐based approach we identified over 1,700 serum proteins with a peptide identification (ID) confidence level of >75%, 605 of which changed significantly between any two patient groups (false discovery rate <5%). Importantly, expression levels of 55 and 15 proteins changed significantly between the simple steatosis and NASH F3/F4 group and the NASH and NASH F3/F4 group, respectively. Classification of proteins with significant changes showed involvement in immune system regulation and inflammation, coagulation, cellular and extracellular matrix structure and function, and roles as carrier proteins in the blood. Further, many of these proteins are synthesized exclusively by the liver and could potentially serve as diagnostic biomarkers for identifying and staging NAFLD. Conclusion: This proteomic analysis reveals important information regarding the pathogenesis/progression of NAFLD and NASH and demonstrates key changes in serum protein expression levels between control subjects and patients with different stages of fatty liver. Future validation of these potential biomarkers is needed such that these proteins may be used in place of liver biopsy to facilitate diagnosis and treatment of patients with NAFLD. (HEPATOLOGY 2009.)

Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer

Purpose: These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. Experimental Design: Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)–positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. Results: ER-positive estradiol-dependent lines (IC50, 12-24 μmol/L) and ER-negative (IC50, 45 μmol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-α levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 ± 8 μmol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (KD, 7.8 μmol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90α short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. Conclusions: Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.