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1.  Perspectives of targeted mass spectrometry for protein biomarker verification 
Current opinion in chemical biology  2009;13(5-6):518-525.
The identification of specific biomarkers will improve the early diagnosis of disease, facilitate the development of targeted therapies, and provide an accurate method to monitor treatment response. A major challenge in the process of verifying biomarker candidates in blood plasma is the complexity and high dynamic range of proteins. This article reviews the current, targeted proteomic strategies that are capable of quantifying biomarker candidates at concentration ranges where biomarkers are expected in plasma (i.e. at the ng/ml level). In addition, a workflow is presented that allows the fast and definitive generation of targeted mass spectrometry-based assays for most biomarker candidate proteins. These assays are stored in publicly accessible databases and have the potential to greatly impact the throughput of biomarker verification studies.
PMCID: PMC2795387  PMID: 19818677
2.  Multi-site assessment of the precision and reproducibility of multiple reaction monitoring–based measurements of proteins in plasma 
Nature biotechnology  2009;27(7):633-641.
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.
PMCID: PMC2855883  PMID: 19561596
3.  Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple-Reaction–Monitoring Mass Spectrometry 
Clinical chemistry  2010;56(12):1804-1813.
If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB).
We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay.
We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay – 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB).
Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability.
PMCID: PMC3103773  PMID: 20923952
4.  Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution*S 
Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1–10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1–10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.
PMCID: PMC2435059  PMID: 17939991
5.  Developing Multiplexed Assays for Troponin I and Interleukin-33 in Plasma by Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry 
Clinical chemistry  2009;55(6):1108-1117.
Protein biomarker candidates from discovery proteomics must be quantitatively verified in patient samples before they can progress to clinical validation. Here we demonstrate that peptide immunoaffinity enrichment coupled with stable isotope dilution mass spectrometry (SISCAPA-MRM) can be used to configure assays with performance suitable for candidate biomarker verification. As proof of principle, we configured SISCAPA assays for troponin I (cTnI), an established biomarker of cardiac injury, and interleukin 33 (IL-33), an emerging immunological and cardiovascular marker for which robust immunoassays are currently not available.
We configured individual and multiplexed assays in which peptides were enriched from digested human plasma using antipeptide antibodies. Assay performance was established using response curves for peptides and proteins spiked into normal plasma. We quantified proteins using labeled peptides as internal standards, and we measured levels of cTnI in patients who underwent a planned myocardial infarction for hypertrophic obstructive cardiomyopathy.
Measurement of cTnI and IL-33 proteins from trypsin-digested plasma was linear from 1.5 to 5000 μg/L, with imprecision <13% for both proteins, processed individually or multiplexed. Results correlated well (R=0.89) with a commercial immunoassay.
We used an established biomarker of cardiac injury and an emerging biomarker to demonstrate how SISCAPA can detect and quantify changes in concentration of proteins present at 1–10 μg/L in plasma. Our results demonstrate that these assays can be multiplexed and retain the necessary precision, reproducibility, and sensitivity to be applied to new and uncharacterized candidate biomarkers for verification of low-abundance proteins in blood.
PMCID: PMC2865473  PMID: 19372185
6.  A pipeline that integrates the discovery and verification of plasma protein biomarkers reveals candidate markers for cardiovascular disease 
Nature Biotechnology  2011;29(7):635-643.
We developed a pipeline to integrate the proteomic technologies used from the discovery to the verification stages of plasma biomarker identification and applied it to identify early biomarkers of cardiac injury from the blood of patients undergoing a therapeutic, planned myocardial infarction (PMI) for treatment of hypertrophic cardiomyopathy. Sampling of blood directly from patient hearts before, during and after controlled myocardial injury ensured enrichment for candidate biomarkers and allowed patients to serve as their own biological controls. LC-MS/MS analyses detected 121 highly differentially expressed proteins, including previously credentialed markers of cardiovascular disease and >100 novel candidate biomarkers for myocardial infarction (MI). Accurate inclusion mass screening (AIMS) qualified a subset of the candidates based on highly specific, targeted detection in peripheral plasma, including some markers unlikely to have been identified without this step. Analyses of peripheral plasma from controls and patients with PMI or spontaneous MI by quantitative multiple reaction monitoring mass spectrometry or immunoassays suggest that the candidate biomarkers may be specific to MI. This study demonstrates that modern proteomic technologies, when coherently integrated, can yield novel cardiovascular biomarkers meriting further evaluation in large, heterogeneous cohorts.
PMCID: PMC3366591  PMID: 21685905
7.  Validation Processes of Protein Biomarkers in Serum—A Cross Platform Comparison 
Sensors (Basel, Switzerland)  2012;12(9):12710-12728.
Due to insufficient biomarker validation and poor performances in diagnostic assays, the candidate biomarker verification process has to be improved. Multi-analyte immunoassays are the tool of choice for the identification and detailed validation of protein biomarkers in serum. The process of identification and validation of serum biomarkers, as well as their implementation in diagnostic routine requires an application of independent immunoassay platforms with the possibility of high-throughput. This review will focus on three main multi-analyte immunoassay platforms: planar microarrays, multiplex bead systems and, array-based surface plasmon resonance (SPR) chips. Recent developments of each platform will be discussed for application in clinical proteomics, principles, detection methods, and performance strength. The requirements for specific surface functionalization of assay platforms are continuously increasing. The reasons for this increase is the demand for highly sensitive assays, as well as the reduction of non-specific adsorption from complex samples, and with it high signal-to-noise-ratios. To achieve this, different support materials were adapted to the immobilized biomarker/ligand, allowing a high binding capacity and immobilization efficiency. In the case of immunoassays, the immobilized ligands are proteins, antibodies or peptides, which exhibit a diversity of chemical properties (acidic/alkaline; hydrophobic/hydrophilic; secondary or tertiary structure/linear). Consequently it is more challenging to develop immobilization strategies necessary to ensure a homogenous covered surface and reliable assay in comparison to DNA immobilization. New developments concerning material support for each platform are discussed especially with regard to increase the immobilization efficiency and reducing the non-specific adsorption from complex samples like serum and cell lysates.
PMCID: PMC3478866  PMID: 23112739
clinical proteomics and diagnostic; multi-analyte immunoassays; serum screening; antibody-antigen interaction
8.  Development of Biomarkers for Screening Hepatocellular Carcinoma Using Global Data Mining and Multiple Reaction Monitoring 
PLoS ONE  2013;8(5):e63468.
Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers and is associated with a poor survival rate. Clinically, the level of alpha-fetoprotein (AFP) has been used as a biomarker for the diagnosis of HCC. The discovery of useful biomarkers for HCC, focused solely on the proteome, has been difficult; thus, wide-ranging global data mining of genomic and proteomic databases from previous reports would be valuable in screening biomarker candidates. Further, multiple reaction monitoring (MRM), based on triple quadrupole mass spectrometry, has been effective with regard to high-throughput verification, complementing antibody-based verification pipelines. In this study, global data mining was performed using 5 types of HCC data to screen for candidate biomarker proteins: cDNA microarray, copy number variation, somatic mutation, epigenetic, and quantitative proteomics data. Next, we applied MRM to verify HCC candidate biomarkers in individual serum samples from 3 groups: a healthy control group, patients who have been diagnosed with HCC (Before HCC treatment group), and HCC patients who underwent locoregional therapy (After HCC treatment group). After determining the relative quantities of the candidate proteins by MRM, we compared their expression levels between the 3 groups, identifying 4 potential biomarkers: the actin-binding protein anillin (ANLN), filamin-B (FLNB), complementary C4-A (C4A), and AFP. The combination of 2 markers (ANLN, FLNB) improved the discrimination of the before HCC treatment group from the healthy control group compared with AFP. We conclude that the combination of global data mining and MRM verification enhances the screening and verification of potential HCC biomarkers. This efficacious integrative strategy is applicable to the development of markers for cancer and other diseases.
PMCID: PMC3661589  PMID: 23717429
9.  Rapid Verification of Candidate Serological Biomarkers Using Gel-based, Label-free Multiple Reaction Monitoring 
Journal of proteome research  2011;10(9):4005-4017.
Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves, quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1-D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μl serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers.
PMCID: PMC3166403  PMID: 21726088
Serum proteomes; serum biomarkers; biomarker verification; biomarker validation; label-free quantitation; multiple reaction monitoring (MRM)
10.  Restructuring proteomics through verification 
Biomarkers in medicine  2010;4(6):799-803.
Proteomics technologies have revolutionized cell biology and biochemistry by providing powerful new tools to characterize complex proteomes, multiprotein complexes and post-translational modifications. Although proteomics technologies could address important problems in clinical and translational cancer research, attempts to use proteomics approaches to discover cancer biomarkers in biofluids and tissues have been largely unsuccessful and have given rise to considerable skepticism. The National Cancer Institute has taken a leading role in facilitating the translation of proteomics from research to clinical application, through its Clinical Proteomic Technologies for Cancer. This article highlights the building of a more reliable and efficient protein biomarker development pipeline that incorporates three steps: discovery, verification and qualification. In addition, we discuss the merits of multiple reaction monitoring mass spectrometry, a multiplex targeted proteomics platform, which has emerged as a potentially promising, high-throughput protein biomarker measurements technology for preclinical ‘verification’.
PMCID: PMC3041639  PMID: 21133699
biomarker; multiple reaction monitoring mass spectrometry; proteomics; verification
11.  Dose-Dependent Proteomic Analysis of Glioblastoma Cancer Stem Cells upon Treatment with Gamma-Secretase Inhibitor 
Proteomics  2011;11(23):4529-4540.
Notch Signaling has been demonstrated to have a central role in Glioblastoma (GBM) Cancer Stem Cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation, a dose-dependent quantitative mass spectrometry (MS) based proteomic study has been performed based on global proteome profiling and a target verification phase where both Immunoassay and a Multiple Reaction Monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation, as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data, possibly due to their crosstalk with Notch Signaling. Overall, this quantitative proteomic based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs.
PMCID: PMC3517080  PMID: 21932445
Glioblastoma; Cancer Stem Cells; Label-free; Multiple Reaction Monitoring; Pathway Analysis
12.  Protein Target Quantification Decision Tree 
The utility of mass spectrometry-(MS-) based proteomic platforms and their clinical applications have become an emerging field in proteomics in recent years. Owing to its selectivity and sensitivity, MS has become a key technological platform in proteomic research. Using this platform, a large number of potential biomarker candidates for specific diseases have been reported. However, due to lack of validation, none has been approved for use in clinical settings by the Food and Drug Administration (FDA). Successful candidate verification and validation will facilitate the development of potential biomarkers, leading to better strategies for disease diagnostics, prognostics, and treatment. With the recent new developments in mass spectrometers, high sensitivity, high resolution, and high mass accuracy can be achieved. This greatly enhances the capabilities of protein biomarker validation. In this paper, we describe and discuss recent developments and applications of targeted proteomics methods for biomarker validation.
PMCID: PMC3562589  PMID: 23401774
13.  SOMAmers and SOMAscan – A Protein Biomarker Discovery Platform for Rapid Analysis of Sample Collections From Bench Top to the Clinic 
Biomarkers are fundamental to nearly every step in the drug discovery and development process, from target validation in the laboratory, to patient stratification in the clinic. Recently, genomic discovery tools have had success in this area, but MS-based proteomic methods less so. This is due to difficulties in developing high throughput assays (ELISA, etc.) for triaging biomarker candidates against large clinical sample collections. However, the SomaLogic proteomics platform is ideally suited to this task. At the heart of the detection technology are SOMAmers (Slow Off-rate Modified Aptamers). They are modified DNA aptamers with high affinity (10∧–9 to 10∧–12 M) and high specificity for their cognate analytes. The assay is highly multiplexed, quantifying >1100 proteins simultaneously from a single 65 uL sample. Sensitivity of the array is generally comparable to sandwich ELISA performance (median LLOQ 100 fM, LoD 40 fM). Samples from a wide variety of sources are amenable to analysis – from serum to CSF, cell/tumor extracts, synovial fluid, etc. Biomarker signatures can be defined in as little as 5 weeks and clinically actionable diagnostics in as little as 6 months. We are currently engaged in a number of clinical discovery applications (Phase 0–4) and have a large number of additional studies completed, in design or sample accrual. The technology and these applications will be discussed.
PMCID: PMC3635296
14.  Mass spectrometry based targeted protein quantification: methods and applications 
Journal of proteome research  2009;8(2):787-797.
The recent advance in technology for mass spectrometry-based targeted protein quantification has opened new avenues for a broad range of proteomic applications in clinical research. The major breakthroughs are highlighted by the capability of using a “universal” approach to perform quantitative assays for a wide spectrum of proteins with minimum restrictions, and the ease of assembling multiplex detections in a single measurement. The quantitative approach relies on the use of synthetic stable isotope labeled peptides or proteins, which precisely mimic their endogenous counterparts and act as internal standards to quantify the corresponding candidate proteins. This report reviews recently developed platform technologies for emerging applications of clinical proteomics and biomarker development.
PMCID: PMC2657955  PMID: 19105742
proteomics; absolute quantification; mass spectrometry; biomarker; MRM; MALDI TOF/TOF; AQUA; SISCAPA; stable isotope dilution
15.  The evolving role of mass spectrometry in cancer biomarker discovery 
Cancer biology & therapy  2009;8(12):1083-1094.
Although the field of mass spectrometry-based proteomics is still in its infancy, recent developments in targeted proteomic techniques have left the field poised to impact the clinical protein biomarker pipeline now more than at any other time in history. For proteomics to meet its potential for finding biomarkers, clinicians, statisticians, epidemiologists and chemists must work together in an interdisciplinary approach. These interdisciplinary efforts will have the greatest chance for success if participants from each discipline have a basic working knowledge of the other disciplines. To that end, the purpose of this review is to provide a nontechnical overview of the emerging/evolving roles that mass spectrometry (especially targeted modes of mass spectrometry) can play in the biomarker pipeline, in hope of making the technology more accessible to the broader community for biomarker discovery efforts. Additionally, the technologies discussed are broadly applicable to proteomic studies, and are not restricted to biomarker discovery.
PMCID: PMC2957893  PMID: 19502776
targeted proteomics; multiple reaction monitoring; selected reaction monitoring; biomarker; mass spectrometry
16.  Coherent pipeline for biomarker discovery using mass spectrometry and bioinformatics 
BMC Bioinformatics  2010;11:437.
Robust biomarkers are needed to improve microbial identification and diagnostics. Proteomics methods based on mass spectrometry can be used for the discovery of novel biomarkers through their high sensitivity and specificity. However, there has been a lack of a coherent pipeline connecting biomarker discovery with established approaches for evaluation and validation. We propose such a pipeline that uses in silico methods for refined biomarker discovery and confirmation.
The pipeline has four main stages: Sample preparation, mass spectrometry analysis, database searching and biomarker validation. Using the pathogen Clostridium botulinum as a model, we show that the robustness of candidate biomarkers increases with each stage of the pipeline. This is enhanced by the concordance shown between various database search algorithms for peptide identification. Further validation was done by focusing on the peptides that are unique to C. botulinum strains and absent in phylogenetically related Clostridium species. From a list of 143 peptides, 8 candidate biomarkers were reliably identified as conserved across C. botulinum strains. To avoid discarding other unique peptides, a confidence scale has been implemented in the pipeline giving priority to unique peptides that are identified by a union of algorithms.
This study demonstrates that implementing a coherent pipeline which includes intensive bioinformatics validation steps is vital for discovery of robust biomarkers. It also emphasises the importance of proteomics based methods in biomarker discovery.
PMCID: PMC2939613  PMID: 20796299
17.  A multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study 
Journal of Proteome Research  2012;11(3):1937-1948.
Biomarkers are most frequently proteins that are measured in the blood. Their development largely relies on antibody creation to test the protein candidate performance in blood samples of diseased versus non-diseased patients. The creation of such antibody assays has been a bottleneck in biomarker progress due to the cost, extensive time and effort required to complete the task. Targeted proteomics is an emerging technology that is playing an increasingly important role to facilitate disease biomarker development. In this study, we applied a SRM-based targeted proteomics platform to directly detect candidate biomarker proteins in plasma to evaluate their clinical utility for pancreatic cancer detection. The characterization of these protein candidates used a clinically well-characterized cohort that included plasma samples from patients with pancreatic cancer, chronic pancreatitis and healthy age-matched controls. Three of the five candidate proteins, including gelsolin, lumican and tissue inhibitor of metalloproteinase 1, demonstrated an AUC value greater than 0.75 in distinguishing pancreatic cancer from the controls. In addition, we provide an analysis of the reproducibility, accuracy, and robustness of the SRM-based proteomics platform. This information addresses important technical issues that could aid in the adoption of the targeted proteomics platform for practical clinical utility.
PMCID: PMC3292708  PMID: 22316387
Targeted proteomics; Mass spectrometer; Selected reaction monitoring (SRM); Multiple reaction monitoring (MRM); Pancreas; Pancreatic ductal adenocarcinoma; Pancreatic cancer; Chronic pancreatitis; Biomarker; Plasma
18.  Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics 
BMC Bioinformatics  2012;13(Suppl 16):S9.
Multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope dilution (SID) is increasingly becoming a widely accepted assay for the quantification of proteins and peptides. These assays have shown great promise in relatively high throughput verification of candidate biomarkers. While the use of MRM-MS assays is well established in the small molecule realm, their introduction and use in proteomics is relatively recent. As such, statistical and computational methods for the analysis of MRM-MS data from proteins and peptides are still being developed. Based on our extensive experience with analyzing a wide range of SID-MRM-MS data, we set forth a methodology for analysis that encompasses significant aspects ranging from data quality assessment, assay characterization including calibration curves, limits of detection (LOD) and quantification (LOQ), and measurement of intra- and interlaboratory precision. We draw upon publicly available seminal datasets to illustrate our methods and algorithms.
PMCID: PMC3489552  PMID: 23176545
Multiple reaction monitoring mass spectrometry (MRM-MS); stable isotope dilution (SID); quantification; interference detection; limits of detection and quantification; intra- and interlaboratory precision
19.  Development of a Chip/Chip/SRM platform using digital chip isoelectric focusing and LC-Chip mass spectrometry for enrichment and quantitation of low abundance protein biomarkers in human plasma 
Journal of proteome research  2011;11(2):808-817.
Protein biomarkers are critical for diagnosis, prognosis, and treatment of disease. The transition from protein biomarker discovery to verification can be a rate limiting step in clinical development of new diagnostics. Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) is becoming an important tool for biomarker verification studies in highly complex biological samples. Analyte enrichment or sample fractionation is often necessary to reduce sample complexity and improve sensitivity of SRM for quantitation of clinically relevant biomarker candidates present at the low ng/mL range in blood. In this paper, we describe an alternative method for sample preparation for LC-SRM MS, which does not rely on availability of antibodies. This new platform is based on selective enrichment of proteotypic peptides from complex biological peptide mixtures via isoelectric focusing (IEF) on a digital ProteomeChip (dPC™) for SRM quantitation using a triple quadrupole (QQQ) instrument with an LC-Chip (Chip/Chip/SRM). To demonstrate the value of this approach, the optimization of the Chip/Chip/SRM platform was performed using prostate specific antigen (PSA) added to female plasma as a model system. The combination of immunodepletion of albumin and IgG with peptide fractionation on the dPC, followed by SRM analysis, resulted in a limit of quantitation of PSA added to female plasma at the level of ~1–2.5 ng/mL with a CV of ~13%. The optimized platform was applied to measure levels of PSA in plasma of a small cohort of male patients with prostate cancer (PCa) and healthy matched controls with concentrations ranging from 1.5 to 25 ng/mL. A good correlation (r2 = 0.9459) was observed between standard clinical ELISA tests and the SRM-based-assay. Our data demonstrate that the combination of IEF on the dPC and SRM (Chip/Chip/SRM) can be successfully applied for verification of low abundance protein biomarkers in complex samples.
PMCID: PMC3656385  PMID: 22098410
Isoelectric focusing; IEF; digital ProteomeChip; dPC; selected reaction monitoring; SRM; prostate specific antigen; PSA; QQQ; LC-Chip
20.  An Extensive Targeted Proteomic Analysis of Disease-Related Protein Biomarkers in Urine from Healthy Donors 
PLoS ONE  2013;8(5):e63368.
The analysis of protein biomarkers in urine is expected to lead to advances in a variety of clinical settings. Several characteristics of urine including a low-protein matrix, ease of testing and a demonstrated proteomic stability offer distinct advantages over current widely used biofluids, serum and plasma. Improvements in our understanding of the urine proteome and in methods used in its evaluation will facilitate the clinical development of urinary protein biomarkers. Multiplexed bead-based immunoassays were utilized to evaluate 211 proteins in urines from 103 healthy donors. An additional 25 healthy donors provided serial urine samples over the course of two days in order to assess temporal variation in selected biomarkers. Nearly one-third of the evaluated biomarkers were detected in urine at levels greater than 1ng/ml, representing a diverse panel of proteins with respect to structure, function and biological role. The presence of several biomarkers in urine was confirmed by western blot. Several methods of data normalization were employed to assess impact on biomarker variability. A complex pattern of correlations with urine creatinine, albumin and beta-2-microglobulin was observed indicating the presence of highly specific mechanisms of renal filtration. Further investigation of the urinary protein biomarkers identified in this preliminary study along with a consideration of the underlying proteomic trends suggested by these findings should lead to an improved capability to identify candidate biomarkers for clinical development.
PMCID: PMC3665773  PMID: 23723977
21.  Computational Biomarker Pipeline from Discovery to Clinical Implementation: Plasma Proteomic Biomarkers for Cardiac Transplantation 
PLoS Computational Biology  2013;9(4):e1002963.
Recent technical advances in the field of quantitative proteomics have stimulated a large number of biomarker discovery studies of various diseases, providing avenues for new treatments and diagnostics. However, inherent challenges have limited the successful translation of candidate biomarkers into clinical use, thus highlighting the need for a robust analytical methodology to transition from biomarker discovery to clinical implementation. We have developed an end-to-end computational proteomic pipeline for biomarkers studies. At the discovery stage, the pipeline emphasizes different aspects of experimental design, appropriate statistical methodologies, and quality assessment of results. At the validation stage, the pipeline focuses on the migration of the results to a platform appropriate for external validation, and the development of a classifier score based on corroborated protein biomarkers. At the last stage towards clinical implementation, the main aims are to develop and validate an assay suitable for clinical deployment, and to calibrate the biomarker classifier using the developed assay. The proposed pipeline was applied to a biomarker study in cardiac transplantation aimed at developing a minimally invasive clinical test to monitor acute rejection. Starting with an untargeted screening of the human plasma proteome, five candidate biomarker proteins were identified. Rejection-regulated proteins reflect cellular and humoral immune responses, acute phase inflammatory pathways, and lipid metabolism biological processes. A multiplex multiple reaction monitoring mass-spectrometry (MRM-MS) assay was developed for the five candidate biomarkers and validated by enzyme-linked immune-sorbent (ELISA) and immunonephelometric assays (INA). A classifier score based on corroborated proteins demonstrated that the developed MRM-MS assay provides an appropriate methodology for an external validation, which is still in progress. Plasma proteomic biomarkers of acute cardiac rejection may offer a relevant post-transplant monitoring tool to effectively guide clinical care. The proposed computational pipeline is highly applicable to a wide range of biomarker proteomic studies.
Author Summary
Novel proteomic technology has led to the generation of vast amounts of biological data and the identification of numerous potential biomarkers. However, computational approaches to translate this information into knowledge capable of impacting clinical care have been lagging. We propose a computational proteomic pipeline for biomarker studies that is founded on the combination of advanced statistical methodologies. We demonstrate our approach through the analysis of data obtained from heart transplant patients. Heart transplantation is the gold standard treatment for patients with end-stage heart failure, but is complicated by episodes of immune rejection that can adversely impact patient outcomes. Current rejection monitoring approaches are highly invasive, requiring a biopsy of the heart. This work aims to reduce the need for biopsies, and demonstrate the power and utility of computational approaches in proteomic biomarker discovery. Our work utilizes novel high-throughput proteomic technology combined with advanced statistical techniques to identify blood markers that guide the decision as to whether a biopsy is warranted, reduce the number of unnecessary biopsies, and ultimately diagnose the presence of rejection in heart transplant patients. Additionally, the proposed computational methodologies can be applied to a range of proteomic biomarker studies of various diseases and conditions.
PMCID: PMC3617196  PMID: 23592955
22.  Quantitative strategies to fuel the merger of discovery and hypothesis-driven shotgun proteomics 
The ultimate goal of most shotgun proteomic pipelines is the discovery of novel biomarkers to direct the development of quantitative diagnostics for the detection and treatment of disease. Differential comparisons of biological samples identify candidate peptides that can serve as proxys of candidate proteins. While these discovery approaches are robust and fairly comprehensive, they have relatively low throughput. When merged with targeted mass spectrometry, this pipeline can fuel hypothesis-driven studies and the development of novel diagnostics and therapeutics.
PMCID: PMC3105289  PMID: 19398505
quantitative shotgun proteomics; biomarker discovery; targeted mass spectrometry; human tissue
23.  Identification of Multiple Novel Protein Biomarkers Shed by Human Serous Ovarian Tumors into the Blood of Immunocompromised Mice and Verified in Patient Sera 
PLoS ONE  2013;8(3):e60129.
The most cancer-specific biomarkers in blood are likely to be proteins shed directly by the tumor rather than less specific inflammatory or other host responses. The use of xenograft mouse models together with in-depth proteome analysis for identification of human proteins in the mouse blood is an under-utilized strategy that can clearly identify proteins shed by the tumor. In the current study, 268 human proteins shed into mouse blood from human OVCAR-3 serous tumors were identified based upon human vs. mouse species differences using a four-dimensional plasma proteome fractionation strategy. A multi-step prioritization and verification strategy was subsequently developed to efficiently select some of the most promising biomarkers from this large number of candidates. A key step was parallel analysis of human proteins detected in the tumor supernatant, because substantially greater sequence coverage for many of the human proteins initially detected in the xenograft mouse plasma confirmed assignments as tumor-derived human proteins. Verification of candidate biomarkers in patient sera was facilitated by in-depth, label-free quantitative comparisons of serum pools from patients with ovarian cancer and benign ovarian tumors. The only proteins that advanced to multiple reaction monitoring (MRM) assay development were those that exhibited increases in ovarian cancer patients compared with benign tumor controls. MRM assays were facilely developed for all 11 novel biomarker candidates selected by this process and analysis of larger pools of patient sera suggested that all 11 proteins are promising candidate biomarkers that should be further evaluated on individual patient blood samples.
PMCID: PMC3609810  PMID: 23544127
24.  Urinary Proteomics to Support Diagnosis of Stroke 
PLoS ONE  2012;7(5):e35879.
Accurate diagnosis in suspected ischaemic stroke can be difficult. We explored the urinary proteome in patients with stroke (n = 69), compared to controls (n = 33), and developed a biomarker model for the diagnosis of stroke. We performed capillary electrophoresis online coupled to micro-time-of-flight mass spectrometry. Potentially disease-specific peptides were identified and a classifier based on these was generated using support vector machine-based software. Candidate biomarkers were sequenced by liquid chromatography-tandem mass spectrometry. We developed two biomarker-based classifiers, employing 14 biomarkers (nominal p-value <0.004) or 35 biomarkers (nominal p-value <0.01). When tested on a blinded test set of 47 independent samples, the classification factor was significantly different between groups; for the 35 biomarker model, median value of the classifier was 0.49 (−0.30 to 1.25) in cases compared to −1.04 (IQR −1.86 to −0.09) in controls, p<0.001. The 35 biomarker classifier gave sensitivity of 56%, specificity was 93% and the AUC on ROC analysis was 0.86. This study supports the potential for urinary proteomic biomarker models to assist with the diagnosis of acute stroke in those with mild symptoms. We now plan to refine further and explore the clinical utility of such a test in large prospective clinical trials.
PMCID: PMC3353991  PMID: 22615742
25.  Clinical proteomic biomarkers: relevant issues on study design & technical considerations in biomarker development 
Biomarker research is continuously expanding in the field of clinical proteomics. A combination of different proteomic–based methodologies can be applied depending on the specific clinical context of use. Moreover, current advancements in proteomic analytical platforms are leading to an expansion of biomarker candidates that can be identified. Specifically, mass spectrometric techniques could provide highly valuable tools for biomarker research. Ideally, these advances could provide with biomarkers that are clinically applicable for disease diagnosis and/ or prognosis. Unfortunately, in general the biomarker candidates fail to be implemented in clinical decision making. To improve on this current situation, a well-defined study design has to be established driven by a clear clinical need, while several checkpoints between the different phases of discovery, verification and validation have to be passed in order to increase the probability of establishing valid biomarkers. In this review, we summarize the technical proteomic platforms that are available along the different stages in the biomarker discovery pipeline, exemplified by clinical applications in the field of bladder cancer biomarker research.
PMCID: PMC3994249  PMID: 24679154
Clinical proteomics; Biomarkers; Verification; Validation; Mass spectrometry

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