A biomarker assay validation approach tailored to the context of use and bioanalytical platform.

It is widely acknowledged by the bioanalytical and biomarker community that biomarker assay validations should be fit-for-purpose depending on the context of use. The challenge is how to consistently apply these principles in teams responsible for measuring a disparate array of biomarkers, often on multiple analytical platforms, at various stages of the drug discovery and development pipeline and across diverse biology focus areas. To drive consistency, while maintaining the necessary flexibility to allow validations to be driven by scientific rationale and taking into consideration the context of use and associated biological and (pre)analytical factors, a framework applicable across biomarker assays was developed. Herein the authors share their perspective to engage in the ongoing conversation around fit-for-purpose biomarker assay validation.

Development and validation of a liquid chromatography-triple quadrupole mass spectrometry method for the determination of isopeptide ε-(γ-glutamyl) lysine in human urine as biomarker for transglutaminase 2 cross-linked proteins.

Determination of the levels of protein cross-linking catalysed by the activity of transglutaminase 2 in various disease states has remained a significant challenge. The ability to quantify the isopeptide ε-(γ-glutamyl) lysine, which can form as a heterogeneous bond within or between proteins has significant analytical and clinical potential as a biomarker in biofluids such as human urine. Increased transglutaminase 2 activity is associated with a number of diseases, such as fibrosis. Previously published methods have been based on classical amino acid analysis, however they require a complex multi-enzyme digestion in order to achieve complete protein digestion, whilst leaving the isopeptide cross link intact. These methods require high levels of enzymes, which contaminate the analysis and alter the dynamics of digestion. The amino acid analysis detection also lacked selectivity, especially where the levels of crosslink are expected to be low relative to the background protein levels. We have systematically addressed these challenges, by optimising the precipitation of the protein in urine, the use of innovative immobilised enzyme technology, which allows for efficient digestion without enzyme contamination and LC-MS/MS detection based on multiple reaction monitoring. This method was validated for its analytical performance characteristics, showing the method has a sensitivity of 0.1 ng/mL of ε-(γ-glutamyl) lysine in human urine with precision of less than 20% CV, and is selective as no interferences were observed that may adversely affect the analysis. As such this approach represents a significant advance in the ability to detect and quantify ε-(γ-glutamyl) lysine.

Relative Quantification of Proteins in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissue Using Multiplexed Mass Spectrometry Assays.

The identification of clinically relevant biomarkers represents an important challenge in oncology. This problem can be addressed with biomarker discovery and verification studies performed directly in tumor samples using formalin-fixed paraffin-embedded (FFPE) tissues. However, reliably measuring proteins in FFPE samples remains challenging. Here, we demonstrate the use of liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM/MS) as an effective technique for such applications. An LC-MRM/MS method was developed to simultaneously quantify hundreds of peptides extracted from FFPE samples and was applied to the targeted measurement of 200 proteins in 48 triple-negative, 19 HER2-overexpressing, and 20 luminal A breast tumors. Quantitative information was obtained for 185 proteins, including known markers of breast cancer such as HER2, hormone receptors, Ki-67, or inflammation-related proteins. LC-MRM/MS results for these proteins matched immunohistochemistry or chromogenic in situ hybridization data. In addition, comparison of our results with data from the literature showed that several proteins representing potential biomarkers were identified as differentially expressed in triple-negative breast cancer samples. These results indicate that LC-MRM/MS assays can reliably measure large sets of proteins using the analysis of surrogate peptides extracted from FFPE samples. This approach allows to simultaneously quantify the expression of target proteins from various pathways in tumor samples. LC-MRM/MS is thus a powerful tool for the relative quantification of proteins in FFPE tissues and for biomarker discovery.

Ontogeny of Small Intestinal Drug Transporters and Metabolizing Enzymes Based on Targeted Quantitative Proteomics.

Most drugs are administered to children orally. An information gap remains on the protein abundance of small intestinal drug-metabolizing enzymes (DMEs) and drug transporters (DTs) across the pediatric age range, which hinders precision dosing in children. To explore age-related differences in DMEs and DTs, surgical leftover intestinal tissues from pediatric and adult jejunum and ileum were collected and analyzed by targeted quantitative proteomics for apical sodium-bile acid transporter, breast cancer resistance protein (BCRP), monocarboxylate transporter 1 (MCT1), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP) 2, MRP3, organic anion-transporting polypeptide 2B1, organic cation transporter 1, peptide transporter 1 (PEPT1), CYP2C19, CYP3A4, CYP3A5, UDP glucuronosyltransferase (UGT) 1A1, UGT1A10, and UGT2B7. Samples from 58 children (48 ileums, 10 jejunums, age range: 8 weeks to 17 years) and 16 adults (8 ileums, 8 jejunums) were analyzed. When comparing age groups, BCRP, MDR1, PEPT1, and UGT1A1 abundance was significantly higher in adult ileum as compared with the pediatric ileum. Jejunal BCRP, MRP2, UGT1A1, and CYP3A4 abundance was higher in the adults compared with children 0-2 years of age. Examining the data on a continuous age scale showed that PEPT1 and UGT1A1 abundance was significantly higher, whereas MCT1 and UGT2B7 abundance was lower in adult ileum as compared with the pediatric ileum. Our data contribute to the deeper understanding of the ontogeny of small intestinal drug-metabolizing enzymes and drug transporters and shows DME-, DT-, and intestinal location-specific, age-related changes. SIGNIFICANCE STATEMENT: This is the first study that describes the ontogeny of small intestinal DTs and DMEs in human using liquid chromatography with tandem mass spectrometry-based targeted quantitative proteomics. The current analysis provides a detailed picture about the maturation of DT and DME abundances in the human jejunum and ileum. The presented results supply age-related DT and DME abundance data for building more accurate PBPK models that serve to support safer and more efficient drug dosing regimens for the pediatric population.

Development of a Highly Multiplexed SRM Assay for Biomarker Discovery in Formalin-Fixed Paraffin-Embedded Tissues.

The search for novel and clinically relevant biomarkers still represents a major clinical challenge and mass-spectrometry-based technologies are essential tools to help in this process. In this application, we demonstrate how selected reaction monitoring (SRM) can be applied in a highly multiplexed way to analyze formalin-fixed paraffin-embedded (FFPE) tissues. Such an assay can be used to analyze numerous samples for narrowing down a list of potential biomarkers to the most relevant candidates. The use of FFPE tissues is of high relevance in this context as large sample collections linked with valuable clinical information are available in hospitals around the world. Here we describe in detail how we proceeded to develop such an assay for 200 proteins in breast tumor FFPE tissues. We cover the selection of suitable peptides, which are different in FFPE compared to fresh frozen tissues and show how we deliberately biased our assay toward proteins with a high probability of being measurable in human clinical samples.

A Novel Positron Emission Tomography (PET) Approach to Monitor Cardiac Metabolic Pathway Remodeling in Response to Sunitinib Malate.

Sunitinib is a tyrosine kinase inhibitor approved for the treatment of multiple solid tumors. However, cardiotoxicity is of increasing concern, with a need to develop rational mechanism driven approaches for the early detection of cardiac dysfunction. We sought to interrogate changes in cardiac energy substrate usage during sunitinib treatment, hypothesising that these changes could represent a strategy for the early detection of cardiotoxicity. Balb/CJ mice or Sprague-Dawley rats were treated orally for 4 weeks with 40 or 20 mg/kg/day sunitinib. Cardiac positron emission tomography (PET) was implemented to investigate alterations in myocardial glucose and oxidative metabolism. Following treatment, blood pressure increased, and left ventricular ejection fraction decreased. Cardiac [18F]-fluorodeoxyglucose (FDG)-PET revealed increased glucose uptake after 48 hours. [11C]Acetate-PET showed decreased myocardial perfusion following treatment. Electron microscopy revealed significant lipid accumulation in the myocardium. Proteomic analyses indicated that oxidative metabolism, fatty acid ß-oxidation and mitochondrial dysfunction were among the top myocardial signalling pathways perturbed. Sunitinib treatment results in an increased reliance on glycolysis, increased myocardial lipid deposition and perturbed mitochondrial function, indicative of a fundamental energy crisis resulting in compromised myocardial energy metabolism and function. Our findings suggest that a cardiac PET strategy may represent a rational approach to non-invasively monitor metabolic pathway remodeling following sunitinib treatment.

Remodeling of plasma lipoproteins in patients with rheumatoid arthritis: Interleukin-6 receptor-alpha inhibition with tocilizumab.

PURPOSE: Rheumatoid arthritis (RA) is associated with increased cardiovascular risk, mediated in part by elevated circulating interleukin-6 levels and proinflammatory changes in plasma lipoproteins. We hypothesized that RA patients acquire inflammation-induced modifications to the protein cargo of circulating lipoproteins that may be reversed by tocilizumab, an interleukin-6 receptor-alpha inhibitor. EXPERIMENTAL DESIGN: Size-exclusion chromatography and reverse-phase protein arrays using 29 antibodies against 26 proteins were applied at baseline and after tocilizumab treatment to analyze the distributions of apolipoproteins, enzymes, lipid transfer proteins, and other associated proteins in plasma lipoprotein fractions from 20 women with RA. RESULTS: A 30% reduction in high-density lipoprotein (HDL)-associated serum amyloid A4 and complement C4 occurred with tocilizumab. Levels of C-reactive protein, associated or comigrating with HDL and low-density lipoprotein (LDL) peaks, were reduced on treatment by approximately 80% and 24%, respectively. Reductions in lipoprotein-associated phospholipase A2, lipoprotein (a), and cholesteryl ester transfer protein in the LDL fraction suggest reductions in LDL-associated proatherogenic factors. Elevations in very low-density lipoprotein (VLDL) enriched with apolipoprotein E were equally observed. CONCLUSIONS AND CLINICAL RELEVANCE: Tocilizumab treatment led to reductions in proinflammatory components and proatherogenic proteins associated with HDL. Whether changes in the proteome of VLDL, LDL, and HDL induced by anti-inflammatory tocilizumab treatment in RA patients modify cardiovascular disease risk requires further investigation.

The Human Autoantibody Response to Apolipoprotein A-I Is Focused on the C-Terminal Helix: A New Rationale for Diagnosis and Treatment of Cardiovascular Disease?

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death worldwide and new approaches for both diagnosis and treatment are required. Autoantibodies directed against apolipoprotein A-I (ApoA-I) represent promising biomarkers for use in risk stratification of CVD and may also play a direct role in pathogenesis. METHODOLOGY: To characterize the anti-ApoA-I autoantibody response, we measured the immunoreactivity to engineered peptides corresponding to the different alpha-helical regions of ApoA-I, using plasma from acute chest pain cohort patients known to be positive for anti-ApoA-I autoantibodies. PRINCIPAL FINDINGS: Our results indicate that the anti-ApoA-I autoantibody response is strongly biased towards the C-terminal alpha-helix of the protein, with an optimized mimetic peptide corresponding to this part of the protein recapitulating the diagnostic accuracy for an acute ischemic coronary etiology (non-ST segment elevation myocardial infarction and unstable angina) obtainable using intact endogenous ApoA-I in immunoassay. Furthermore, the optimized mimetic peptide strongly inhibits the pathology-associated capacity of anti-ApoA-I antibodies to elicit proinflammatory cytokine release from cultured human macrophages. CONCLUSIONS: In addition to providing a rationale for the development of new approaches for the diagnosis and therapy of CVD, our observations may contribute to the elucidation of how anti-ApoA-I autoantibodies are elicited in individuals without autoimmune disease.

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues.

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.

Identification of six cell surface proteins for specific liver targeting.

PURPOSE: Cell surface proteins are the primary means for a cell to sense and interact with its environment and their dysregulation has been linked to numerous diseases. In particular, the identification of proteins specific to a single tissue type or to a given disease phenotype may enable the characterization of novel therapeutic targets. We tested here the feasibility of a cell surface proteomics approach to identify pertinent markers directly in a clinically relevant tissue. EXPERIMENTAL DESIGN: We analyzed the cell surface proteome of freshly isolated primary heptatocytes using a glycocapture-specific approach combined with a robust bioinformatics filtering. RESULTS: Using primary lung epithelial cell cultures as negative controls, we identified 32 hepatocyte-specific cell surface proteins candidates. We used mRNA expression to select six markers that may provide adequate specificity for targeting therapeutics to the liver. CONCLUSIONS AND CLINICAL RELEVANCE: We demonstrate the feasibility and the importance of conducting such studies directly in a clinically relevant tissue. In particular, the cell surface proteome of freshly isolated hepatocytes differed substantially from cultured cell lines.

Proteomics in pharmaceutical research and development.

In the 20 years since its inception, the evolution of proteomics in pharmaceutical industry has mirrored the developments within academia and indeed other industries. From initial enthusiasm and subsequent disappointment in global protein expression profiling, pharma research saw the biggest impact when relating to more focused approaches, such as those exploring the interaction between proteins and drugs. Nowadays, proteomics technologies have been integrated in many areas of pharmaceutical R&D, ranging from the analysis of therapeutic proteins to the monitoring of clinical trials. Here, we review the development of proteomics in the drug discovery process, placing it in a historical context as well as reviewing the current status in light of the contributions to this special issue, which reflect some of the diverse demands of the drug and biomarker pipelines.

Characterization of a human pluripotent stem cell-derived model of neuronal development using multiplexed targeted proteomics.

PURPOSE: Human pluripotent stem cell (hPSC)-derived cellular models have great potential to enable drug discovery and improve translation of preclinical insights to the clinic. We have developed a hPSC-derived neural precursor cell model for studying early events in human brain development. We present protein-level characterization of this model, using a multiplexed SRM approach, to establish reproducibility and physiological relevance; essential prerequisites for utilization of the neuronal development model in phenotypic screening-based drug discovery. EXPERIMENTAL DESIGN: Profiles of 246 proteins across three key stages of in vitro neuron differentiation were analyzed by SRM. Three independently hPSC-derived isogenic neural stem cell (NSC) lines were analyzed across five to nine independent neuronal differentiations. RESULTS: One hundred seventy-five proteins were reliably quantified revealing a time-dependent pattern of protein regulation that reflected protein dynamics during in vivo brain development and that was conserved across replicate differentiations and multiple cell lines. CONCLUSIONS AND CLINICAL RELEVANCE: SRM-based protein profiling enabled establishment of the reproducibility and physiological relevance of the hPSC-derived neuronal model. Combined with the successful quantification of proteins relevant to neurodevelopmental diseases, this validates the platform for use as a model to enable neuroscience drug discovery.

The role of quantitative ADME proteomics to support construction of physiologically based pharmacokinetic models for use in small molecule drug development.

Pharmacokinetics (PK) refers to the time course of drug concentrations in the body and since knowledge of PK aids understanding of drug efficacy and safety, numerous PK studies are performed in animals and humans during the drug development process. In vitro to in vivo extrapolation and physiologically based pharmacokinetic (PBPK) modeling are tools that integrate data from various in silico, in vitro, and in vivo sources to deliver mechanistic quantitative simulations of in vivo PK. PBPK models are used to predict human PK and to evaluate the effects of intrinsic factors such as organ dysfunction, age, and genetics as well as extrinsic factors such as co-administered drugs. In recent years, the use of PBPK within the industry has greatly increased. However, insufficient data on how the abundance of metabolic enzymes and membrane transporters vary in different human patient populations and in different species has been a limitation. A major advance is therefore expected through reliable quantification of the abundance of these proteins in tissues. This review describes the role of PBPK modeling in drug discovery and development, outlines the assumptions involved in integrating protein abundance data, and describes the advances made and expected in determining abundance of relevant proteins through mass spectrometric techniques.

Quantitative ADME proteomics - CYP and UGT enzymes in the Beagle dog liver and intestine.

PURPOSE: Beagle dogs are used to study oral pharmacokinetics and guide development of drug formulations for human use. Since mechanistic insight into species differences is needed to translate findings in this species to human, abundances of cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) drug metabolizing enzymes have been quantified in dog liver and intestine. METHODS: Abundances of enzymes were measured in Beagle dog intestine and liver using selected reaction monitoring mass spectrometry. RESULTS: Seven and two CYPs were present in the liver and intestine, respectively. CYP3A12 was the most abundant CYP in both tissues. Seven UGT enzymes were quantified in the liver and seven in the intestine although UGT1A11 and UGT1A9 were present only in the intestine and UGT1A7 and UGT2B31 were found only in the liver. UGT1A11 and UGT1A2 were the most abundant UGTs in the intestine and UGT2B31 was the most abundant UGT in the liver. Summed abundance of UGT enzymes was similar to the sum of CYP enzymes in the liver whereas intestinal UGTs were up to four times more abundant than CYPs. The estimated coefficients of variation of abundance estimates in the livers of 14 donors were separated into biological and technical components which ranged from 14 to 49% and 20 to 39%, respectively. CONCLUSIONS: Abundances of canine CYP enzymes in liver and intestine have been confirmed in a larger number of dogs and UGT abundances have been quantified for the first time. The biological variability in hepatic CYPs and UGTs has also been estimated.

Biomarkers for cardiovascular risk assessment in autoimmune diseases.

Autoimmune diseases, such as antiphospholipid syndrome, systemic lupus erythematosus, and rheumatoid arthritis, are characterized by a high prevalence of cardiovascular (CV) disease (CVD), which constitutes the leading causes of morbidity and mortality among such patients. Although such effects are partly explained by a higher prevalence of traditional CV risk factors, many studies indicate that such factors do not fully explain the enhanced CV risk in these patients. In addition, risk stratification algorithms based upon traditional CV risk factors are not as predictive in autoimmune diseases as in the general population. For these reasons, the timely and accurate assessment of CV risk in these high-risk populations still remains an unmet clinical need. An enhanced contribution of different inflammatory components of the immune response, as well as autoimmune elements (e.g. autoantibodies, autoantigens, and cellular response), has been proposed to underlie the incremental CV risk observed in these populations. Recent advances in proteomic tools have contributed to the discovery of proteins involved in CVDs, including some that may be suitable to be used as biological markers. In this review we summarize the main markers in the field of CVDs associated with autoimmunity, as well as the recent advances in proteomic technology and their application for biomarker discovery in autoimmune disease.

Definition of human apolipoprotein A-I epitopes recognized by autoantibodies present in patients with cardiovascular diseases.

Autoantibodies to apolipoprotein A-I (anti-apoA-I IgG) have been shown to be both markers and mediators of cardiovascular disease, promoting atherogenesis and unstable atherosclerotic plaque. Previous studies have shown that high levels of anti-apoA-I IgGs are independently associated with major adverse cardiovascular events in patients with myocardial infarction. Autoantibody responses to apoA-I can be polyclonal and it is likely that more than one epitope may exist. To identify the specific immunoreactive peptides in apoA-I, we have developed a set of methodologies and procedures to isolate, purify, and identify novel apoA-I endogenous epitopes. First, we generated high purity apoA-I from human plasma, using thiophilic interaction chromatography followed by enzymatic digestion specifically at lysine or arginine residues. Immunoreactivity to the different peptides generated was tested by ELISA using serum obtained from patients with acute myocardial infarction and high titers of autoantibodies to native apoA-I. The immunoreactive peptides were further sequenced by mass spectrometry. Our approach successfully identified two novel immunoreactive peptides, recognized by autoantibodies from patients suffering from myocardial infarction, who contain a high titer of anti-apoA-I IgG. The discovery of these epitopes may open innovative prognostic and therapeutic opportunities potentially suitable to improve current cardiovascular risk stratification.

Automated sample preparation platform for mass spectrometry-based plasma proteomics and biomarker discovery.

The identification of novel biomarkers from human plasma remains a critical need in order to develop and monitor drug therapies for nearly all disease areas. The discovery of novel plasma biomarkers is, however, significantly hampered by the complexity and dynamic range of proteins within plasma, as well as the inherent variability in composition from patient to patient. In addition, it is widely accepted that most soluble plasma biomarkers for diseases such as cancer will be represented by tissue leakage products, circulating in plasma at low levels. It is therefore necessary to find approaches with the prerequisite level of sensitivity in such a complex biological matrix. Strategies for fractionating the plasma proteome have been suggested, but improvements in sensitivity are often negated by the resultant process variability. Here we describe an approach using multidimensional chromatography and on-line protein derivatization, which allows for higher sensitivity, whilst minimizing the process variability. In order to evaluate this automated process fully, we demonstrate three levels of processing and compare sensitivity, throughput and reproducibility. We demonstrate that high sensitivity analysis of the human plasma proteome is possible down to the low ng/mL or even high pg/mL level with a high degree of technical reproducibility.

Applications of mass spectrometry for quantitative protein analysis in formalin-fixed paraffin-embedded tissues.

Proteomic analysis of tissues has advanced in recent years as instruments and methodologies have evolved. The ability to retrieve peptides from formalin-fixed paraffin-embedded tissues followed by shotgun or targeted proteomic analysis is offering new opportunities in biomedical research. In particular, access to large collections of clinically annotated samples should enable the detailed analysis of pathologically relevant tissues in a manner previously considered unfeasible. In this paper, we review the current status of proteomic analysis of formalin-fixed paraffin-embedded tissues with a particular focus on targeted approaches and the potential for this technique to be used in clinical research and clinical diagnosis. We also discuss the limitations and perspectives of the technique, particularly with regard to application in clinical diagnosis and drug discovery.

Determination of free desmosine in human plasma and its application in two experimental medicine studies.

Elastin is one of the major extracellular matrix proteins associated with connective tissue. Its degradation leads to the liberation of the unique amino acids desmosine and isodesmosine. These have shown utility as biomarkers of elastin breakdown for disease progression, patient stratification, and drug efficacy. So far, the quantitation of desmosines in plasma is hampered by complex sample preparation. Here we demonstrate an improved and simplified procedure for detecting both free and total desmosines. The method is based on spiking with a deuterium-labeled desmosine standard, ethanol precipitation, propionylation, high-performance liquid chromatography (HPLC) separation, and selected reaction monitoring (SRM) mass spectrometry. The performance of the assay is illustrated by comparing the levels of free and total desmosines in normal healthy plasma and those from patients diagnosed with chronic obstructive pulmonary disease (COPD). A conserved ratio of 1:3 for free to total desmosine was found. The determination of free desmosine has higher accuracy than that of total desmosine; therefore, it is the method of choice when plasma volume is limiting. Finally, we show that the plasma desmosine concentration correlates with age and body mass index.