A pipeline that integrates the discovery and verification of plasma protein biomarkers reveals candidate markers for cardiovascular disease

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
Nature Biotechnology (Impact Factor: 41.51). 06/2011; 29(7):635-43. DOI: 10.1038/nbt.1899
Source: PubMed


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.

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    • "The total cost-of-data for MSIA was <$11.25, with unit costs of less than $10 per analysis, and fixed costs of approximately $1.25. Other MS-based approaches typically include more involved sample preparation before analysis and longer times devoted to data acquisition using LC/MS(MS), which lengthens the time-to-data, reduces throughput and increases costs (e.g., a 15-minute LC/MS run at $150/hour [32] equates to ∼$40 in fixed costs on top of any time needed for sample preparation and unit costs). However, the cost-of-data (as well as time-to-data and throughput) demonstrated here is on par, or better than those of current FDA-Approved IGF1 ELISA approaches (e.g., IDS-iSYS). "
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    ABSTRACT: Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories. Presented here is an MS-based quantitative IGF1 assay with performance rating of >1,000 samples/day, and a capability of quantifying IGF1 point mutations and posttranslational modifications. The throughput of the IGF1 mass spectrometric immunoassay (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a tip format, and high-throughput MALDI-TOF MS analysis. The Limit of Detection and Limit of Quantification of the resulting assay were 1.5 μg/L and 5 μg/L, respectively, with intra- and inter-assay precision CVs of less than 10%, and good linearity and recovery characteristics. The IGF1 MSIA was benchmarked against commercially available IGF1 ELISA via Bland-Altman method comparison test, resulting in a slight positive bias of 16%. The IGF1 MSIA was employed in an optimized parallel workflow utilizing two pipetting robots and MALDI-TOF-MS instruments synced into one-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data collection, and data processing. Using this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This rate of assaying is a significant improvement over existing MS-based IGF1 assays, and is on par with that of the enzyme-based immunoassays. Furthermore, a mutation was detected in ∼1% of the samples (SNP: rs17884626, creating an A→T substitution at position 67 of the IGF1), demonstrating the capability of IGF1 MSIA to detect point mutations and posttranslational modifications.
    PLoS ONE 03/2014; 9(3):e92801. DOI:10.1371/journal.pone.0092801 · 3.23 Impact Factor
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    • "The large number of putative protein biomarkers that exist in the literature needs to be validated, something which requires high precision, robust assays with high throughput, appropriate for the analysis of thousands of patient samples. Quantitative mass spectrometric assays, like MRM (multiple reaction monitoring), have a huge potential in both the validation of newly discovered biomarker candidates and in the measurements of known markers within a multiplex assay format in the clinical routine [2] [3] [4] [5] [6]. Thanks to the SISCAPA (stable isotope standards and capture by antipeptide antibodies ) technique [7] [8] [9], the sensitivity of mass spectrometry based methods is not a limiting factor anymore in comparison with the classical immune-based assays; nevertheless the capability to multiplexing has enormous advantage. "

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    • "Claims of predictive importance obviously need to be validated in longitudinal follow-up studies [8]. Attempts have been made to streamline biomarker development by establishing a pipeline from initial discovery to clinical application [9] [10]. Such a pipeline is intended to include the successive steps required to establish new biomarkers, along with a framework of analytical and statistical methods that can be used at each step to avoid the problems discussed above through proper study design. "
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    ABSTRACT: The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PLA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.
    Biochimica et Biophysica Acta 08/2013; 1844(5). DOI:10.1016/j.bbapap.2013.07.016 · 4.66 Impact Factor
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