Notable peer reviewed articles–MS-based immunoassays (MSIA) as an alternative to enzyme-linked immunosorbent assays (ELISA) for protein quantification.
Articles of note
Hybrid immunoaffinity–mass spectrometric methods for efficient protein biomarker verification in pharmaceutical development (full free text)
In the discovery and development of novel protein biomarkers, mass spectrometry (MS) and ligand-binding (LB) assays have traditionally operated at different ends of a common continuum. Liquid chromatography (LC)–MS/MS, with its ability to simultaneously measure thousands of peptides in a single run, has driven the development of proteomics, one of the primary tools used in the search for novel protein biomarkers. LB assays, on the other hand, have been applied to the quantitative analysis of discrete proteins and are the method of choice for clinical diagnostic assays. In simple terms, LC–MS/MS has been primarily used for hypothesis generation, while LB methods have typically been reserved for hypothesis testing. Interestingly, with the trend towards targeted multiprotein analysis (using either MS or LB) growing, the protein biomarker landscape has changed, meaning the roles of these methodologies are far less well defined. As discussed in this article, a positive outcome of this research is the development of hybrid immunoaffinity (IA)–MS methods, which hold significant importance for the discovery and development of novel protein biomarkers. Learn more ›
Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum (free full text)
OBJECTIVES: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum.
DESIGN AND METHODS: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. Learn more ›
Mass spectrometry-based (MS) methods are effective tools for discovering protein biomarker candidates that can differentiate between physiological and pathophysiological states. Promising candidates are validated in studies comprising large patient cohorts. Here, targeted protein analytics are used to increase sample throughput. Methods involving antibodies, such as sandwich immunoassays or Western blots, are commonly applied at this stage. Highly-specific and sensitive mass spectrometry-based immunoassays that have been established in recent years offer a suitable alternative to sandwich immunoassays for quantifying proteins. Learn more ›
Evaluation of large scale quantitative proteomic assay development using peptide affinity-based mass spectrometry (free full text)
Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. Learn more ›
Mass spectrometric immunoassay and MRM as targeted MS-based quantitative approaches in biomarker development: potential applications to cardiovascular disease and diabetes
Type 2 diabetes (T2DM) is an important risk factor for cardiovascular disease (CVD)—the leading cause of death in the US. Yet not all subjects with T2DM are at equal risk for CVD complications; the challenge lies in identifying those at greatest risk. Therapies directed towards treating conventional risk factors have failed to significantly reduce this residual risk in T2DM patients. Thus newer targets and markers are needed for the development and testing of novel therapies. Herein we review two complementary mass spectrometry-based approaches—Mass Spectrometric Immunoassay (MSIA) and tandem mass spectrometry as multiple reaction monitoring (MRM)—for the analysis of plasma proteins and post translational modifications (PTMs) of relevance to T2DM and CVD. Together, these complementary approaches allow for high-throughput monitoring of many PTMs and the absolute quantification of proteins near the low picomolar range. Learn more ›
Mass spectrometric immunoassay of intact insulin and related variants for population proteomics studies (free full text)
PURPOSE: The purpose of the work presented herein was to develop a high-throughput assay for the quantification of human insulin in plasma samples while simultaneously detecting, with high mass accuracy, any additional variant forms of insulin that might be present in each sample.
EXPERIMENTAL DESIGN: A mass spectrometric immunoassay (MSIA) was designed in which anti-human insulin antibodies were immobilized to commercially available mass spectrometric immunoassay pipette tips and used to capture insulin and related protein variants from human plasma. Learn more ›
Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)
A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, alpha1 antichymotrypsin, interleukin-6, and tumor necrosis factor-alpha) from a pool of 10,203 in silico tryptic peptide candidates representing 237 known plasma components. Learn more ›
Targeted selected reaction monitoring mass spectrometric immunoassay for insulin-like growth factor 1 (free full text)
Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. Learn more ›
Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers
The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. Learn more ›
Mass spectrometric immunoassay revisited (free full text)
The progressive understanding and improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), realized over the years through the considerable efforts of Dr. Marvin Vestal, have made possible numerous comparable efforts involving its application in the biological sciences. Here we revisit the concepts behind one such analytical approach, Mass Spectrometric Immunoassay, which is designed to selectively detect and quantify proteins present in biological milieu. Learn more ›
Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants (free full text)
BACKGROUND: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminal PTH variants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84.
METHODS: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards. Learn more ›
BACKGROUND: Retinol Binding Protein 4 (RBP4) is an exciting new biomarker for the determination of insulin resistance and type 2 diabetes. It is known that circulating RBP4 resides in multiple variants which may provide enhanced clinical utility, but conventional immunoassay methods are blind to such differences. A Mass Spectrometric immunoassay (MSIA) technology that can quantitate total RBP4 as well as individual isoforms may provide an enhanced analysis for this biomarker.
METHODS: RBP4 was isolated and detected from 0.5 uL of human plasma using MSIA technology, for the simultaneous quantification and differentiation of endogenous human RBP4 and its variants. Learn more ›