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.
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.
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 μL 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.
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.
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 A1c), 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.
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