Protein Biomarker Assays Research Articles

Recommendations for Selection and Characterization of Protein Biomarker Assay Calibrator Material

As biomarkers continue to become an integral part of drug development and decision-making, there are increased expectations for reliable and quantitative assays. Protein biomarker assay results are directly influenced by the calibrator material. The selection of calibrator material presents many challenges that impact the relative accuracy and performance of the assay. There is an industry-wide challenge finding reliable and well-characterized calibrator material with good documentation. Several case studies are presented that demonstrate some of the challenges involved in selecting appropriate calibrators along with the resolutions that were ultimately applied. From these experiences, we present here a set of recommendations for selecting and characterizing calibrator material based on the intended purpose of the assay. Finally, we introduce a commutability approach, based on common clinical chemistry practices, which can be used to demonstrate inter-changeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.

Large-scale implementation of sequential protein and peptide immunoaffinity enrichment LC/nanoLC-MS/MS for human β-nerve growth factor.

A previously described immunoaffinity (IA)-LC-MS/MS assay for human β-nerve growth factor (β-NGF) was implemented to support large-scale sample testing for multiple clinical trials. Methodology & results: The procedure was modified to increase throughput by simultaneous preparation of two 96-well plates and LC duty-cycle reduction. Robustness of the LC method and nano-ESI was ensured during large-scale assay execution by closely monitoring and, if needed, replacing system components prior to failure. Following validation, the assay was used to analyze approximately 19,000 samples from multiple clinical studies over several years. Routine implementation of the β-NGF IA-LC-MS/MS assay supported drug development programs. This optimized assay format now serves as a template for other clinical protein biomarker assays.

Age-related variations: A retrospective analysis of 851 prospectively collected patient samples to determine the benefit of combining combinatorial protein biomarker assay for risk assessment in women with dense breast.

27 Background: Clinicians often experience difficulty in differentiating benign lesions from invasive breast cancers in patients designated as dense breast. A major limitation of radiological breast cancer screening methods involves a decrease in sensitivity and specificity in women with dense breast. Thus, we sought to test whether Klarify Breast, a combinatorial protein-based biomarker panel could improve early detection of significant breast lesions in a controlled fashion. Clearly, a diagnostic assay that would provide biochemical evidence in the patient’s clinical course is greatly needed. Methods: We have conducted two independent, multi-center, prospective clinical trials to establish the clinical validity of Klarify Breast – an assay that uses multiple Serum Protein Biomarkers (SPBs), Tumor-Associated Autoantibodies (TAAbs), patient specific clinical data and develop a score to differentiate patients with benign breast disease from those with invasive breast cancer. Independent panels of biomarkers and associated algorithms were developed using prospectively collected samples from women under age 50 (n = 351) and from women ages 25-75 (n = 500). Here, we present the benefit of integrating the results of Klarify Breast test with patient imaging to best assess risk in women with dense breast. Results: While performance of the assay was somewhat age dependent (women under the age of 50 demonstrated a higher sensitivity and specificity than women over the age of 50). Here we present data that both groups of women clearly benefit from the addition of a biomarker assay combined with standard of care imaging in identifying invasive breast lesions. Conclusions: Clearly, in women with dense breast, where radiologic studies alone do not permit full assessment in women with a dense breast finding. The biomarker test here, comprised of TAAbs and SPBs, offers a clear advantage in terms of NPV, PPV, Sensitivity and specificity. The results argue strongly for the use of appropriate biomarkers to augment imaging based breast cancer screening in women with dense breast or those who are at high risk. Clinical trial information: NCT01839045, NCT02078570.

Systems for multiplexing homogeneous immunoassays.

High-throughput multiplex protein biomarker assays continue to gain significance in the fields of biomarker discovery and drug development, due to their economical use of not only the precious clinical biological samples but also expensive reagents. Among these platforms, homogeneous multiplex systems have potential for short assay run times and cost-effective reagent consumptions. However, these systems must overcome challenges of signal cross talk and biochemical cross-reactivity. Despite these obstacles, several homogeneous multiplex immunoassays have been demonstrated. These include fluorescent polarization, fluorescent resonance energy transfer with quantum dots or graphene, luminescent oxygen-channeling immunoassay coupled with aqueous two-phase systems and DNA proximity assays. The balance between speed/simplicity and high multiplexing and robustness of these homogeneous multiplex immunoassays are discussed in this review.

Early biomarkers of joint damage in rheumatoid and psoriatic arthritis.

Joint destruction, as evidenced by radiographic findings, is a significant problem for patients suffering from rheumatoid arthritis and psoriatic arthritis. Inherently irreversible and frequently progressive, the process of joint damage begins at and even before the clinical onset of disease. However, rheumatoid and psoriatic arthropathies are heterogeneous in nature and not all patients progress to joint damage. It is therefore important to identify patients susceptible to joint destruction in order to initiate more aggressive treatment as soon as possible and thereby potentially prevent irreversible joint damage. At the same time, the high cost and potential side effects associated with aggressive treatment mean it is also important not to over treat patients and especially those who, even if left untreated, would not progress to joint destruction. It is therefore clear that a protein biomarker signature that could predict joint damage at an early stage would support more informed clinical decisions on the most appropriate treatment regimens for individual patients. Although many candidate biomarkers for rheumatoid and psoriatic arthritis have been reported in the literature, relatively few have reached clinical use and as a consequence the number of prognostic biomarkers used in rheumatology has remained relatively static for several years. It has become evident that a significant challenge in the transition of biomarker candidates to clinical diagnostic assays lies in the development of suitably robust biomarker assays, especially multiplexed assays, and their clinical validation in appropriate patient sample cohorts. Recent developments in mass spectrometry-based targeted quantitative protein measurements have transformed our ability to rapidly develop multiplexed protein biomarker assays. These advances are likely to have a significant impact on the validation of biomarkers in the future. In this review, we have comprehensively compiled a list of candidate biomarkers in rheumatoid and psoriatic arthritis, evaluated the evidence for their potential as biomarkers of bone (joint) damage, and outlined how mass spectrometry-based targeted and multiplexed measurement of candidate biomarker proteins is likely to accelerate their clinical validation and the development of clinical diagnostic tests.

Development of microcolumn-based one-site immunometric assays for protein biomarkers

One-site immunometric assays that utilize affinity microcolumns were developed and evaluated for the analysis of protein biomarkers. This approach used labeled antibodies that were monitored through on-line fluorescence or near-infrared (NIR) fluorescence detection. Human serum albumin (HSA) was utilized as a model target protein for this approach. In these assays, a fixed amount of labeled anti-HSA antibodies was mixed with samples or standards containing HSA, followed by the injection of this mixture onto an HSA microcolumn to remove excess antibodies and detect the non-retained labeled antibodies that were bound to HSA from the sample. The affinity microcolumns were 2.1mm i.d. ×5mm and contained 8–9nmol of immobilized HSA. These microcolumns were used from 0.10 to 1.0mL/min and gave results within 35s to 2.8min of sample injection. Limits of detection down to 0.10–0.28ng/mL (1.5–4.2pM) or 25–30pg/mL (0.38–0.45pM) were achieved when using fluorescein or a NIR fluorescent dye as the label, with an assay precision of ±0.1–4.2%. Several parameters were examined during the optimization of these assays, and general guidelines and procedures were developed for the extension of this approach for use with other types of affinity microcolumns and protein biomarkers.

Integration of analytical techniques for protein biomarker assays.

Integration of analytical techniques for protein biomarker assays.

MRM validation of targeted nonglycosylated peptides from N-glycoprotein biomarkers using direct trypsin digestion of undepleted human plasma

A rapid, simple, and reproducible MRM-based validation method for serological glycoprotein biomarkers in clinical use was developed by targeting the nonglycosylated tryptic peptides adjacent to N-glycosylation sites. Since changes in protein glycosylation are known to be associated with a variety of diseases, glycoproteins have been major targets in biomarker discovery. We previously found that nonglycosylated tryptic peptides adjacent to N-glycosylation sites differed in concentration between normal and hepatocellular carcinoma (HCC) plasma due to differences in steric hindrance of the glycan moiety in N-glycoproteins to tryptic digestion (Lee et al., 2011). To increase the feasibility and applicability of clinical validation of biomarker candidates (nonglycosylated tryptic peptides), we developed a method to effectively monitor nonglycosylated tryptic peptides from a large number of plasma samples and to reduce the total analysis time with maximizing the effect of steric hindrance by the glycans during digestion of glycoproteins. The AUC values of targeted nonglycosylated tryptic peptides were excellent (0.955 for GQYCYELDEK, 0.880 for FEDGVLDPDYPR and 0.907 for TEDTIFLR), indicating that these could be effective biomarkers for hepatocellular carcinoma. This method provides the necessary throughput required to validate glycoprotein biomarkers, as well as quantitative accuracy for human plasma analysis, and should be amenable to clinical use. Difficulties in verifying and validating putative protein biomarkers are often caused by complex sample preparation procedures required to determine their concentrations in a large number of plasma samples. To solve the difficulties, we developed MRM-based protein biomarker assays that greatly reduce complex, time-consuming, and less reproducible sample pretreatment steps in plasma for clinical implementation. First, we used undepleted human plasma samples without any enrichment procedures. Using nanoLC/MS/MS, we targeted nonglycosylated tryptic peptides adjacent to N-linked glycosylation sites in N-linked glycoprotein biomarkers, which could be detected in human plasma samples without depleting highly abundant proteins. Second, human plasma proteins were digested with trypsin without reduction and alkylation procedures to minimize sample preparation. Third, trypsin digestion times were shortened so as to obtain reproducible results with maximization of the steric hindrance effect of the glycans during enzyme digestion. Finally, this rapid and simple sample preparation method was applied to validate targeted nonglycosylated tryptic peptides as liver cancer biomarker candidates for diagnosis in 40 normal and 41 hepatocellular carcinoma (HCC) human plasma samples. This strategy provided the necessary throughput required to monitor protein biomarkers, as well as quantitative accuracy in human plasma analysis. From biomarker discovery to clinical implementation, our method will provide a biomarker study platform that is suitable for clinical deployment, and can be applied to high-throughput approaches.

Quantitative mass spectrometry in proteomics

Investigating living systems at the protein level is continuing to provide important insights into many biological processes across all kingdoms of life. Mass spectrometry based proteomics has fundamentally changed the way in which biological systems are interrogated because of its ability to measure thousands of proteins and post-translational modifications in parallel. This enables investigations at all levels of biological complexity ranging from protein complexes to human patient populations. While five years ago, the majority of proteomic experiments mostly enumerated the protein constituents of a biological system, quantitative measurements are at the heart of practically every proteomic study today. This shift was once more significantly driven by developments in mass spectrometry which is now the de-facto standard for quantitative measurements in proteomics. Alongside, the miniaturization of associated sample preparation and separation techniques as well as the adoption and further development of data analysis strategies from other –omics research have helped transform proteomics from a qualitative to a quantitative science. At the same time, the areas of life science research to which proteomic methods are being applied are expanding rapidly. While a few years ago, most of the successful proteomic work was performed in rather simple models such as unicellular organisms or cell lines derived of animal and plant origin, it is becoming increasingly possible to work with primary samples including that of human patients. However, scientists engaged in such studies had to realize that the enormous molecular complexity and the dynamic nature of proteomes posed much larger challenges than those encountered for either genome or transcriptome studies. In particular, issues related to splice variants, post-translational modifications, dynamic range of protein expression spanning 10 orders of magnitude, protein stability, transient protein associations, and dependence on cell type or physiological state etc. are still limiting our technical ability to characterize proteomes comprehensively and reproducibly in a reasonable time. Unsurprisingly, numerous experimental strategies and schemes have been devised to address the many challenges and, as in any developing field, some have come and gone, while others have been adopted more broadly. It is probably safe to assume that we have not yet seen the end of the technical developments which, in turn, will allow more and more difficult biological systems to be approached by proteomics. Despite all the hurdles, mass spectrometry based quantitative proteomics increasingly impacts life science research in many areas including protein expression profiling, the analysis of signaling pathways or the development of protein biomarker assay systems to name a few. It is important to note that in each area, distinct scientific questions are being asked and, therefore, distinct proteomic approaches will have to be applied, many of which vary widely in their versatility, technical maturity, difficulty, and expense. Consequently, we must recognize that some scientific questions are, and will remain, much harder to answer by proteomics than others. Still, the strong trend towards the alternative scientific paradigm of system-level interrogation of biological phenomena is unstoppable and will without doubt continue to provide important complementary views to traditional and still highly successful hypothesis-driven research. In this special issue on quantitative mass spectrometry in proteomics, we have assembled a group of reputed scientists who all have significant statements to make regarding the topic of this issue. There are eight reviews that provide an in-depth appreciation of a state-of-the-art technology alongside six original research articles highlighting some of the current trends. We can of course not claim to cover the field comprehensively, but do believe that the content of this special issue has a lot to Published in the topical issue Quantitative Mass Spectrometry in Proteomics with guest editors Bernhard Kuster and Marcus Bantscheff.

Abstract 5114: Mass spectrometric immunoassays for quantitative determination of protein biomarker variants

Abstract Post-translational modifications and genetic variations give rise to protein variants that significantly increase human proteome complexity. Modified proteins also play an important role in biological processes. Mass spectrometry, coupled to immunoaffinity separations, provides an efficient mean for simultaneous detection and quantification of protein variants. In Mass Spectrometric Immunoassays, affinity interactions are utilized to selectively retrieve specific proteins from complex biological fluids, and are followed by mass spectrometric analysis of the targeted proteins. These assays are similar to traditional enzyme immunoassays in that they use antibodies as reagents for affinity retrieval of specific proteins, but instead of enzymatic reaction for indirect protein detection, mass spectrometric analysis is used for direct identification of the proteins and their modifications. Described here are mass spectrometric immunoassays for targeted quantitative proteomics analysis of specific protein modifications. Assays for several protein biomarkers exhibiting posttranslational modifications were developed, including beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin. The following assay performance characteristics were examined (and determined): LOQs (sufficient for quantification of normal/healthy levels of the proteins from human serum or plasma); intra- and inter-assay precision (CVs of <10%), linearity and spiking recovery (90-110% range); specificity (<5% cross reactivity with other proteins), and correlation with ELISA-generated results (acceptable Passing & Bablok fits and Cusum linearity p-values of >0.1). The new assays were then utilized to determine the individual concentration of the specific protein variants across larger cohorts of samples (n=500), demonstrating the ability to fully quantify individual forms of post-translationally modified proteins. The mass spectrometric immunoassays are ideally suited for detection and quantification of novel protein variants, not known a priori. These MS-based assays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5114. doi:10.1158/1538-7445.AM2011-5114

Challenges to Developing Proteomic-Based Breast Cancer Diagnostics

Over the past decade, multiple genetic and histological approaches have accelerated development of new breast cancer diagnostics and treatment paradigms. Multiple distinct genetic subtypes of breast cancers have been defined, and this has progressively led toward more personalized medicine in regard to treatment options. There still remains a deficiency in the development of molecular diagnostic assays that can be used for breast cancer detection and pretherapy clinical decisions. In particular, the type of cancer-specific biomarker typified by a serum or tissue-derived protein. Progress in this regard has been minimal, especially in comparison to the rapid advancements in genetic and histological assays for breast cancers. In this review, some potential reasons for this large gap in developing protein biomarkers will be discussed, as well as new strategies for improving these approaches. Improvements in the study design of protein biomarker discovery strategies in relation to the genetic subtypes and histology of breast cancers is also emphasized. The current successes in use of genetic and histological assays for breast cancer diagnostics are summarized, and in that context, the current limitations of the types of breast cancer-related clinical samples available for protein biomarker assay development are discussed. Based on these limitations, research strategies emphasizing identification of glycoprotein biomarkers in blood and MALDI mass spectrometry imaging of tissues are described.