Cory E. Bystrom

Plasma renin activity by LC-MS/MS: development of a prototypical clinical assay reveals a subpopulation of human plasma samples with substantial peptidase activity.

BACKGROUND For management and treatment of secondary hypertension, plasma renin activity (PRA) assay is considered an essential diagnostic tool. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to PRA offering improvements in laboratory workflow and throughput. During development, we observed a substantial number of clinical samples that have strong degradation activity toward angiotensin (Ang) I during generation. A preliminary characterization of this degradation activity was performed, and we provide here a method by which this degradation can be monitored via the addition of an isotope-labeled degradation standard. METHODS Automated online sample extraction coupled with HPLC was used to isolate Ang I and internal standard from plasma. The effluent from the analytical column was directed to a triple quadrupole MS operated in selected reaction monitoring mode, monitoring the a(5) and b(5) product ions from the [M+3H](+3) precursors. Routine analysis could be achieved with as little as 150 μL plasma. RESULTS We identified both C-terminal and N-terminal degradation products of Ang I using isotope-labeled peptides as controls and substrates. In 2%-5% of patient samples, the degradation essentially eliminated any Ang I produced during generation. CONCLUSIONS Our method requires reduced sample handling when compared with an RIA and eliminates the need for extended generation times for samples with low renin activity. Degradation of Ang I during generation appears to be a confounding variable in the interpretation of results from some clinical samples. Samples with profound degradation activity can be identified using a degradation standard that is added at the start of generation.

Narrow Mass Extraction of Time-of-Flight Data for Quantitative Analysis of Proteins: Determination of Insulin-Like Growth Factor-1

Methods for quantitative analysis of proteins by mass spectrometry have progressed dramatically. While isotope-dilution approaches using selected reaction monitoring of tryptic peptides (also known as bottom up) have become common, the potential to use narrow mass extraction of high-resolution mass spectra provides a compelling alternative. We investigated the relationships between instrument performance and data processing with the aim of determining whether this approach can lead to robust bioanalytical assays for proteins. Our approach utilized off-line sample preparation combined with online sample extraction coupled to HPLC with the effluent from the analytical column directed to a high-resolution, high-mass accuracy quadrupole time-of-flight (qTOF) mass spectrometer operated in full scan mode. Narrow mass extraction of a single isotope from IGF-1 in the 7+ charge state (m/z 1093.5209) was used to generate extracted ion chromatograms. We found that with appropriate attention to instrument performance and data processing, quantitative protein assays with good sensitivity, high selectivity, and excellent analytical performance can be developed.

Clinical Utility of Insulin-Like Growth Factor 1 and 2; Determination by High Resolution Mass Spectrometry

Measurement of insulin-like growth factor-1 (IGF-I) has utility for the diagnosis and management of growth disorders, but inter-assay comparison of results has been complicated by a multitude of reference standards, antibodies, detection methods, and pre-analytical preparation strategies. We developed a quantitative LC-MS method for intact IGF-I, which has advantages in throughput and complexity when compared to mass spectrometric approaches that rely on stable isotope dilution analysis of tryptic peptides. Since the method makes use of full-scan data, the assay was easily extended to provide quantitative measurement of IGF-II using the same assay protocol. The validated LC-MS assay for IGF-I and IGF-II provides accurate results across the pediatric and adult reference range and is suitable for clinical use.

Measurement of Lipoprotein-Associated Phospholipase A2 by Use of 3 Different Methods: Exploration of Discordance between ELISA and Activity Assays

BACKGROUND Lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme associated with inflammation, is used as a biomarker for cardiovascular disease risk. Both the concentration and activity of Lp-PLA2 have been shown to be clinically relevant. However, there is a discordance between the serum concentration of Lp-PLA2 measured by the standard ELISA-based immunoassays and the activity of this enzyme, leading to substantial discordance in risk categorization depending on assay format. METHODS We developed 2 LC-MS/MS-based assays to quantify serum Lp-PLA2 activity (multiple reaction monitoring detection of product) and concentration [stable isotope standards and capture by antipeptide antibody (SISCAPA) immunoaffinity], and we investigated their correlation to commercially offered colorimetric activity and immunometric concentrations assays. Associations between Lp-PLA2 and lipoproteins and the effect of selected detergents in liberating Lp-PLA2 were evaluated by use of immunoprecipitation and Western blot analyses. RESULTS Serum Lp-PLA2 concentrations measured by quantitative SISCAPA-mass spectrometry were substantially higher than concentrations typically measured by immunoassay and showed an improved agreement with Lp-PLA2 activity. With detergents, liberation of Lp-PLA2 from lipoprotein complexes dramatically increased the amount of protein detected by immunoassay and improved the agreement with activity measurements. CONCLUSIONS Quantitative analysis of Lp-PLA2 concentration and activity by LC-MS/MS assays provided key insight into resolving the well-documented discordance between Lp-PLA2 concentration (determined by immunoassay) and activity. Quantitative detection of Lp-PLA2 by immunoassay appears to be strongly inhibited by interaction of Lp-PLA2 with lipoprotein. Together, the results illustrate the advantages of quantitative LC-MS/MS for measurement of Lp-PLA2 concentration (by SISCAPA) and activity (by direct product detection).

The Analysis of Native Proteins and Peptides in the Clinical Lab Using Mass Spectrometry

Mass spectrometric analysis of proteins and peptides has now enjoyed more than 2 decades of vigorous development and growth. As a key analytical technology that has been used with great success for the analysis of small molecules, the development of mass spectrometry instrumentation, software, and informatics tools in support of proteomics research has set the stage for protein and peptide analysis to emerge in the clinical lab. This transition is aided by an emphasis on translational research wherein one aim is the focused conversion of laboratory discoveries into useful clinical tools. Mass spectrometry (MS) is now being driven into service in the clinical laboratory as both a research tool and analytical platform on which accurate and precise diagnostic measurements of proteins and peptides can be made. Mass spectrometry is not a newcomer to the clinical lab. The implementation of metabolite screening for inborn errors of metabolism is a tremendous success story and established the credibility of mass spectrometry in the diagnostics field in the early 1990’s. 1 Since that time, many commercial and hospital labs have adopted MS tools for the quantitative analysis of therapeutic drugs, drugs of abuse, and steroids. 2,3 The benefits of these approaches have been well documented, with improved selectivity, precision and accuracy affording improved clinical utility of diagnostic results. In addition, mass spectrometry-based assays can readily be automated, providing improvements in productivity compared to manual assays that are difficult to implement on robotic platforms. While the analysis of polypeptides in the clinical lab has traditionally been performed using immunochemical techniques, it appears that mass spectrometry is poised to make significant inroads in this area by bringing a new level of

A Step Toward Simplicity for a Complex Analyte

α1-Antitrypsin (A1AT)2 deficiency is a serious, life-threatening disorder driven by abnormally low circulating concentrations of A1AT, which lead to protease–antiprotease imbalance. This imbalance accentuates protease activity and can lead to tissue damage, the most important of which leads to impairment of lung function (1). The apparent simplicity of diagnosing this disorder by a single measurement of the A1AT concentration is belied by the variety of A1AT tests offered by every reference laboratory. Physicians frequently evaluate genotyping, phenotyping, and concentration measurements in toto or through results produced via an algorithm that involves reflexing from one test to another (2). The need for this complex approach reflects the intricate relationships between genotype, phenotype, protein function, and concentration. The challenge in arriving at appropriate diagnosis and treatment comes from the variable penetrance of A1AT genetic defects and the limitations of each test. Typical genotyping tests detect only the most common disease alleles, S and Z, unless full exon sequencing is used. Phenotyping by isoelectric focusing can detect known and unknown polymorphs, but only in cases in which the amino acid substitution leads to a change in the proteins isoelectric point. Finally, A1AT is an acute-phase protein, and circulating concentrations …