Mass Spectrometric Immunoassay for the qualitative and quantitative analysis of the cytokine Macrophage Migration Inhibitory Factor (MIF).

BACKGROUND: The cytokine MIF (Macrophage Migration Inhibitory Factor) has diverse physiological roles and is present at elevated concentrations in numerous disease states. However, its molecular heterogeneity has not been previously investigated in biological samples. Mass Spectrometric Immunoassay (MSIA) may help elucidate MIF post-translational modifications existing in vivo and provide additional clarity regarding its relationship to diverse pathologies. RESULTS: In this work, we have developed and validated a fully quantitative MSIA assay for MIF, and used it in the discovery and quantification of different proteoforms of MIF in serum samples, including cysteinylated and glycated MIF. The MSIA assay had a linear range of 1.56-50 ng/mL, and exhibited good precision, linearity, and recovery characteristics. The new assay was applied to a small cohort of human serum samples, and benchmarked against an MIF ELISA assay. CONCLUSIONS: The quantitative MIF MSIA assay provides a sensitive, precise and high throughput method to delineate and quantify MIF proteoforms in biological samples.

Examining serum amyloid P component microheterogeneity using capillary isoelectric focusing and MALDI-MS.

Serum amyloid P component (SAP) is a glycoprotein of interest due to its presence in amyloid plaque formations. As with most glycoproteins, SAP can possibly vary greatly in its isoforms, which can be an important factor toward understanding the role of SAP. Interestingly, previous characterizations suggest varying degrees of microheterogeneity, some of which are in conflict. In this work, we provide new information to clarify SAP's microheterogeneity profile using CIEF to carefully analyze pooled samples and by studying individual samples across populations with mass spectrometric immunoassay. With respect to CIEF, a single pI band was observed suggesting that human SAP does not have extensive heterogeneity concluded from gel IEF experiments in the past. Additionally, this is supported by a population study, which revealed an overwhelming degree of uniformity. Overall, this work corroborates the idea that SAP is relatively consistent across the population and with respect to microheterogeneity.

Building multidimensional biomarker views of type 2 diabetes on the basis of protein microheterogeneity.

BACKGROUND: In 2008, the US Food and Drug Administration (FDA) issued a Guidance for Industry statement formally recognizing (during drug development) the conjoined nature of type 2 diabetes (T2D) and cardiovascular disease (CVD), which has precipitated an urgent need for panels of markers (and means of analysis) that are able to differentiate subtypes of CVD in the context of T2D. Here, we explore the possibility of creating such panels using the working hypothesis that proteins, in addition to carrying time-cumulative marks of hyperglycemia (e.g., protein glycation in the form of Hb A(1c)), may carry analogous information with regard to systemic oxidative stress and aberrant enzymatic signaling related to underlying pathobiologies involved in T2D and/or CVD. METHODS: We used mass spectrometric immunoassay to quantify, in targeted fashion, relative differences in the glycation, oxidation, and truncation of 11 specific proteins. RESULTS: Protein oxidation and truncation (owing to modified enzymatic activity) are able to distinguish between subsets of diabetic patients with or without a history of myocardial infarction and/or congestive heart failure where markers of glycation alone cannot. CONCLUSION: Markers based on protein modifications aligned with the known pathobiologies of T2D represent a reservoir of potential cardiovascular markers that are needed to develop the next generation of antidiabetes medications.

Intrapersonal and populational heterogeneity of the chemokine RANTES.

BACKGROUND: Current immunoassays for the chemokine RANTES (regulated on activation, normal T-cell expressed and secreted) are not tailored for specific isoforms that exist endogenously, despite the fact that variants with modified activity are known to exist. This is surprising in view of this protein's ubiquitous increased presence in many diseases and that the 2 established isoforms are truncated by enzymes also correlated to disease. An in-depth population survey of RANTES heterogeneity in the context of multiple diseases via a mass spectrometric immunoassay (MSIA) may resolve this issue. METHODS: We developed an MSIA for RANTES and endogenous variants apparent in human plasma. Samples from multiple cohorts of individuals (type 2 diabetes, congestive heart failure, history of myocardial infarction, and cancer patients) were run in parallel with samples from healthy individuals (239 people total). We used 230 microL of plasma per individual and tabulated relative percent abundance (RPA) values for identified isoforms. RESULTS: We detected at least 19 variants, including the dipeptidyl peptidase IV (DPP-IV)-truncated variant. The majority of variants were unreported in the literature. Identifiable modifications included N- and/or C-terminal truncations, oxidation, glycation, and glycosylation. We observed statistically significant differences in RPA values for multiple variants between disease cohorts and recognized prospective disease-specific protein profiles for RANTES. CONCLUSIONS: Because of widespread interest in the clinical value of RANTES, the protein diversity established here may aid in the design of future, fully quantitative assays. Equally important, an inclusive qualitative understanding of RANTES heterogeneity may present new insights into the relationship between RANTES and disease.

Full-length characterization of proteins in human populations.

BACKGROUND: Diversity in human proteins often gives rise to pluralities of structurally similar but functionally distinct proteins. Such microheterogeneity generally escapes proteomics discovery technologies, as well as conventional immunometric assays. As an intermediate between these 2 technological approaches, targeted, full-length characterization of proteins using mass spectrometry is a suitable means of defining microheterogeneity evident in human populations. CONTENT: We describe and explore the implications of microheterogeneity using the exemplar of human vitamin D binding protein (Gc-Globulin) as observed in cohorts of 400 individuals. Our investigations yielded: (a) population frequency data comparable to genotyping; (b) population frequency data for protein variants, with and without genotype linkage; (c) reference values for the different protein variants per cohort and genotype; and (d) associations between variant, frequency, relative abundance, and diseases. SUMMARY: With the exception of the genotype frequency, such population data are unique and illustrate a need to more fully understand the exact full-length qualitative and quantitative idiosyncrasies of individual proteins in relation to health and disease as part of the standardized biomarker development and clinical proteomic investigation of human proteins.

Parallel workflow for high-throughput (>1,000 samples/day) quantitative analysis of human insulin-like growth factor 1 using mass spectrometric immunoassay.

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.

Mass spectrometric immunoassay of intact insulin and related variants for population proteomics studies.

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. RESULTS: Standard curves for insulin exhibited linearity (average R(2) for three days of analysis=0.99) and assay concentration limits of detection and limits of quantification for insulin were found to be 1 and 15 pM, respectively. Estimated coefficient of variations for inter-day experiments (n=3 days) were <8%. Simultaneously, the assay was shown to detect and identify insulin metabolites and synthetic insulin analogs (e.g. Lantus). Notably, insulin variants not known to exist in plasma were detected in diabetics. CONCLUSIONS AND CLINICAL RELEVANCE: This introductory study sets a foundation toward the screening of large populations to investigate insulin isoforms, isoform frequencies, and their quantification.

C-peptide microheterogeneity in type 2 diabetes populations.

PURPOSE: The purpose of this study was to investigate naturally occurring C-peptide microheterogeneity in healthy and type 2 diabetes (T2D) populations. EXPERIMENTAL DESIGN: MS immunoassays capable of simultaneously detecting intact C-peptide and variant forms were applied to plasma samples from 48 healthy individuals and 48 individuals diagnosed with T2D. RESULTS: Common throughout the entire sample set were three previously unreported variations of C-peptide. The relative contribution of one variant, subsequently identified as C-peptide (3-31), was found to be more abundant in the T2D population as compared to the healthy population. Dipeptidyl peptidase IV is suspected to be responsible for this particular cleavage product, which is consistent with the pathophysiology of T2D. CONCLUSIONS AND CLINICAL RELEVANCE: C-peptide does not exist in the human body as a single molecular species. It is qualitatively more heterogeneous than previously thought. These results lay a foundation for future studies devoted to a comprehensive understanding of C-peptide and its variants in healthy and diabetic populations.

Population studies of intact vitamin D binding protein by affinity capture ESI-TOF-MS.

Blood plasma proteins with molecular weights greater than approximately 30 kDa are refractory to comprehensive, high-throughput qualitative characterization of microheterogeneity across human populations. Analytical techniques for obtaining high mass resolution for targeted, intact protein characterization and, separately, high sample throughput exist, but efficient means of coupling these assay characteristics remain rather limited. This article discusses the impetus for analyzing intact proteins in a targeted manner across populations and describes the methodology required to couple mass spectrometric immunoassay with electrospray ionization mass spectrometry for the purpose of qualitatively characterizing a prototypical large plasma protein, vitamin D binding protein, across populations.

Population studies of Vitamin D Binding Protein microheterogeneity by mass spectrometry lead to characterization of its genotype-dependent O-glycosylation patterns.

Mass spectrometric evidence presented here characterizes the genotype-dependent glycosylation patterns for each of the three major allele products of Vitamin D Binding Protein found in the general human population. Findings based on the analysis of over 100 individual plasma samples demonstrated that all DBP allele products, except GC*2, are modified (10-25 mol%) with a linear (NeuNAc) 1(Gal) 1(GalNAc) 1 trisaccharide and, to a much lesser extent (1-5 mol%) with a trisaccharide-independent (Gal) 1(GalNAc) 1 dissaccharide. GC*2 protein contains the disaccharide but remains completely free of the trisaccharide, even in heterozygous individuals possessing a second gene product that is modified with the trisaccharide. Thus, all allelic forms of DBP except GC*2 possess two independent O-glycosylation sites occupied by separate, yet consistently isomass oligosaccharides and, despite a consensus sequence, lack N-glycosylation.