Leigh Anderson

An Automated and Multiplexed Method for High Throughput Peptide Immunoaffinity Enrichment and Multiple Reaction Monitoring Mass Spectrometry-based Quantification of Protein Biomarkers

There is an urgent need for quantitative assays in verifying and validating the large numbers of protein biomarker candidates produced in modern “-omics” experiments. Stable isotope standards with capture by anti-peptide antibodies (SISCAPA) has shown tremendous potential to meet this need by combining peptide immunoaffinity enrichment with quantitative mass spectrometry. In this study, we describe three significant advances to the SISCAPA technique. First, we develop a method for an automated magnetic bead-based platform capable of high throughput processing. Second, we implement the automated method in a multiplexed SISCAPA assay (nine targets in one assay) and assess the performance characteristics of the multiplexed assay. Using the automated, multiplexed platform, we demonstrate detection limits in the physiologically relevant ng/ml range (from 10 μl of plasma) with sufficient precision (median coefficient of variation, 12.6%) for quantifying biomarkers. Third, we demonstrate that enrichment of peptides from larger volumes of plasma (1 ml) can extend the limits of detection to the low pg/ml range of protein concentration. The method is generally applicable to any protein or biological specimen of interest and holds great promise for analyzing large numbers of biomarker candidates.

Protein Quantitation through Targeted Mass Spectrometry : The Way Out of Biomarker Purgatory?

The enormous potential of biomarkers to revolutionize clinical practice and improve patient care has been well documented (1)(2). Molecular-based diagnostic and prognostic tests, particularly those aimed at protein analytes, could be used to detect disease earlier, enabling treatment to start sooner and possibly cure rather than to merely delay further injury or death. These tools could also be used to stage disease more accurately and to predict response to therapy, thereby helping to select the correct treatment. Biomarkers can also be used to stratify patients for the assessment of new drug therapies and to serve as surrogate endpoints in early-phase drug trials, thereby lowering the overall cost of drug development and producing more effective treatments. Given their high potential therapeutic and financial impacts, that so few new protein biomarkers have been introduced into widespread clinical use recently is, on the surface, surprising. In fact, only 5 new protein markers have been approved by the US Food and Drug Administration in the last 5 years for measurement in plasma or serum \[the information on protein markers in Anderson and Anderson, 2002, has been updated with information from the Center for Devices and Radiological Health, US Food and Drug Administration\] (3)(4). The reasons for the dearth of new protein biomarkers are gradually becoming clearer. They are related to the high false-discovery rate of “omics” methods (regardless of the technology used), combined with a lack of robust methods for biomarker verification in large clinical sample sets (5)(6)(7)(8). It is now common for differential analyses of tissue or plasma samples by multidimensional liquid chromatography–tandem mass spectrometry (LC-MS/MS)1 (the workhorse tool for unbiased discovery) to confidently identify thousands of proteins, hundreds of which can vary in concentration by 5-fold or more between case …

Analytical techniques for cell fractions. Xxviii. Dissection of complex antigenic mixtures using monoclonal antibodies and two-dimensional gel electrophoresis.

Abstract Disposable microimmunoadsorbent columns containing Staphylococcus Protein A and monoclonal antibodies were used to bind antigenic proteins from a mixture. Eluates from these columns were directly analyzed by electrophoresis on two-dimensional (2-D) gels. In this way, biochemical and biophysical information on the bound antigen and on the specific antibody can be obtained simultaneously. The microimmunoadsorbents are easy to handle and in conjunction with multiple 2-D gel systems provide a means for screening large numbers of myeloma hybrids for specificity to antigens in complex mixtures.

11 – Use of Large-Scale Two-Dimensional ISODALT Gel Electrophoresis System in Immunology

Publisher Summary This chapter focuses on the usage of the large-scale two-dimensional ISODALT gel electrophoresis system in immunology. At Argonne National Laboratory, the ISODALT system, where ISO describes the charge separation and DALT stands for size separation, is a large, multipurpose operation. The chapter also describes one version of the ISODALT system adapted at the Basel Institute for Immunology for specific problems occurring in immunology. In 1975, O’Farrell developed an analytical technique for the resolution of complex protein mixture using a combination of two separation procedures—charge separation and size separation. The electrophoretograms obtained by this technique allow a fine comparison of the protein pattern within a single run. The final aim is to perform a comparison using a computer, the function of which is not only to compare two gels adjusted by matching, stretching, and fitting but also to analyze gels by comparing the presence or absence of individual spots or constellations of spots with a cumulative experimental data file.

Within Sight of a Rational Pipeline for Development of Protein Diagnostics

Given the paucity of new clinical tests emerging from proteomics research on the one hand (1) and the large and increasing levels of NIH funding devoted to biomarker research on the other (2), important questions have been raised about our strategy for translating basic research into clinical diagnostics. At a fundamental level, it has been unclear whether the lack of clinical results from numerous biomarker discovery efforts is due to our having exhausted the low-hanging fruit (or all fruit?) in years past, or to defects in the approaches being used in the search for new markers. It seems unlikely that all useful clinical protein tests have been discovered; therefore, discussion has focused on the defects in the “biomarker pipeline” that should be conveying candidate biomarker proteins toward a clinical application. The ultimate importance of this debate is not academic: Improvements in clinical care and in basic health economics increasingly depend on the development of new tests (including companion diagnostics that allow personalization of therapy) and markers addressing the long-standing list of unmet clinical diagnostic needs in such areas as Alzheimer disease, chronic obstructive pulmonary disease, and cancer. Fortunately, recent publications (3, 4) suggest that progress is being made in implementing biomarker pipeline concepts that work: approaches that link a “discovery” process that uses a small number of particularly favorable samples with one or more “qualification” and/or “verification” steps that focus on testing the resulting candidate biomarkers with independent sets of real clinical samples. The work of Addona et al. (3) serves as a useful benchmark in this context because it is focused on a clinical indication (cardiac damage markers) in which numerous proteins, such as creatine kinase isoenzyme MB, troponins, and B-type natriuretic peptide, have achieved undisputed clinical success. One can thus ask whether such a modern systematic approach …