Disialylated apolipoprotein C-III proteoform is associated with improved lipids in prediabetes and type 2 diabetes.

The apoC-III proteoform containing two sialic acid residues (apoC-III2) has different in vitro effects on lipid metabolism compared with asialylated (apoC-III0) or the most abundant monosialylated (apoC-III1) proteoforms. Cross-sectional and longitudinal associations between plasma apoC-III proteoforms (by mass spectrometric immunoassay) and plasma lipids were tested in two randomized clinical trials: ACT NOW, a study of pioglitazone in subjects with impaired glucose tolerance (n = 531), and RACED (n = 296), a study of intensive glycemic control and atherosclerosis in type 2 diabetes patients. At baseline, higher relative apoC-III2 and apoC-III2/apoC-III1 ratios were associated with lower triglycerides and total cholesterol in both cohorts, and with lower small dense LDL in the RACED. Longitudinally, changes in apoC-III2/apoC-III1 were inversely associated with changes in triglycerides in both cohorts, and with total and small dense LDL in the RACED. apoC-III2/apoC-III1 was also higher in patients treated with PPAR-γ agonists and was associated with reduced cardiovascular events in the RACED control group. Ex vivo studies of apoC-III complexes with higher apoC-III2/apoC-III1 showed attenuated inhibition of VLDL uptake by HepG2 cells and LPL-mediated lipolysis, providing possible functional explanations for the inverse association between a higher apoC-III2/apoC-III1 and hypertriglyceridemia, proatherogenic plasma lipid profiles, and cardiovascular risk.

The association of plasma cystatin C proteoforms with diabetic chronic kidney disease.

BACKGROUND: Cystatin C (CysC) is an endogenous cysteine protease inhibitor that can be used to assess the progression of kidney function. Recent studies demonstrate that CysC is a more specific indicator of glomerular filtration rate (GFR) than creatinine. CysC in plasma exists in multiple proteoforms. The goal of this study was to clarify the association of native CysC, CysC missing N-terminal Serine (CysC des-S), and CysC without three N-terminal residues (CysC des-SSP) with diabetic chronic kidney disease (CKD). RESULTS: Using mass spectrometric immunoassay, the plasma concentrations of native CysC and the two CysC truncation proteoforms were examined in 111 individuals from three groups: 33 non-diabetic controls, 34 participants with type 2 diabetes (DM) and without CKD and 44 participants with diabetic CKD. Native CysC concentrations were 1.4 fold greater in CKD compared to DM group (p = 0.02) and 1.5 fold greater in CKD compared to the control group (p = 0.001). CysC des-S concentrations were 1.55 fold greater in CKD compared to the DM group (p = 0.002) and 1.9 fold greater in CKD compared to the control group (p = 0.0002). CysC des-SSP concentrations were 1.8 fold greater in CKD compared to the DM group (p = 0.008) and 1.52 fold greater in CKD compared to the control group (p = 0.002). In addition, the concentrations of CysC proteoforms were greater in the setting of albuminuria. The truncated CysC proteoform concentrations were associated with estimated GFR independent of native CysC concentrations. CONCLUSION: Our findings demonstrate a greater amount of CysC proteoforms in diabetic CKD. We therefore suggest assessing the role of cystatin C proteoforms in the progression of CKD.

Development of quantitative mass spectrometric immunoassay for serum amyloid A.

OBJECTIVE: Proteins can exist as multiple proteoforms in vivo that can have important roles in physiological and pathological states. METHODS: We present the development and characterization of mass spectrometric immunoassay (MSIA) for quantitative determination of serum amyloid A (SAA) proteoforms. RESULTS: Intra- and inter-day precision revealed CVs <10%. Against existing SAA ELISA, the developed MSIA showed good correlation according to the Altman-Bland plot. Individual concentrations of the SAA proteoforms across a cohort of 170 samples revealed 7 diverse SAA polymorphic types and 12 different proteoforms. CONCLUSION: The new SAA MSIA enables parallel analysis of SAA polymorphisms and quantification of all expressed SAA proteoforms, in a high-throughput and time-efficient manner.

Development of multiplex mass spectrometric immunoassay for detection and quantification of apolipoproteins C-I, C-II, C-III and their proteoforms.

The impetus for discovery and evaluation of protein biomarkers has been accelerated by recent development of advanced technologies for rapid and broad proteome analyses. Mass spectrometry (MS)-based protein assays hold great potential for in vitro biomarker studies. Described here is the development of a multiplex mass spectrometric immunoassay (MSIA) for quantification of apolipoprotein C-I (apoC-I), apolipoprotein C-II (apoC-II), apolipoprotein C-III (apoC-III) and their proteoforms. The multiplex MSIA assay was fast (∼ 40 min) and high-throughput (96 samples at a time). The assay was applied to a small cohort of human plasma samples, revealing the existence of multiple proteoforms for each apolipoprotein C. The quantitative aspect of the assay enabled determination of the concentration for each proteoform individually. Low-abundance proteoforms, such as fucosylated apoC-III, were detected in less than 20% of the samples. The distribution of apoC-III proteoforms varied among samples with similar total apoC-III concentrations. The multiplex analysis of the three apolipoproteins C and their proteoforms using quantitative MSIA represents a significant step forward toward better understanding of their physiological roles in health and disease.

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.

RNA-protein binding interface in the telomerase ribonucleoprotein.

Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR-TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA-protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain.

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.

Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants.

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. RESULTS: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28-84, aa48-84, aa34-77, aa37-77, and aa38-77. Quantitative SRM assays were developed for PTH1-84, PTH7-84, and variant aa34-84. Peptides exhibited linear responses (R(2) = 0.90-0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16-31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%-12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%-9%. CONCLUSIONS: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.

Dependence of the ejection velocities of laser-ablated ions on the laser wavelength and fluence.

Drift measurements of initial ejection velocities of matrix-assisted laser desorption/ionization matrix compounds have been made as a function of ablating laser wavelength and laser fluence. For pulsed laser irradiation just above the matrix ion appearance threshold, initial ejection velocities of protonated molecular ions of an anthranilic acid target increase from ~ 1350 m/s to ~ 1640 m/s (kinetic energies of 1.3 eV and 1.9 eV, respectively) when the ablation laser wavelength is changed from 355 nm to 266 nm. Increasing the laser fluence per pulse by up to a factor of 10 above threshold results in the appearance of a slower component of the ejected ion flux. The results are interpreted by a photomechanical ejection model in which a photoexcited surface molecule instantaneously becomes larger and recoils away from the surface.

Top-down mass spectrometric immunoassay for human insulin and its therapeutic analogs.

Measurement of insulin and its therapeutic analogs is important in diabetes, hypoglycemia, sports anti-doping and toxicology. Commercial insulin immunoassays fail to detect commonly prescribed insulin analogs. Because of their unique sequences and masses, these analogs are readily measured and distinguished with mass spectrometric (MS) assays. Reviewed here is an insulin mass spectrometric immunoassay (MSIA) that combines micro-scale immunoaffinity capture with sensitive MS detection of insulin and its therapeutic analogs. An antibody reactive to all insulin analogs was used to affinity capture the insulin analogs. Following elution, insulins were detected with MALDI-TOF MS or LC-MS analysis. Isotopic resolution for insulin was achieved for both MS techniques, and several insulin analogs were detected at unique m/z signals. Porcine insulin, spiked in all samples, served as an internal reference standard for quantification. Linear standard curves spanning three orders of magnitude were obtained, with limits of detection of 15pM for the MALDI-TOF MS and 1.5pM for the LC-MS. This insulin assay was capable of detecting and quantifying not only human endogenous insulin, but also most of the therapeutic insulin analogs, which could find use in diagnosis of severe hypoglycemia and in sports anti-doping. SIGNIFICANCE: Insulin replacement therapy consists of injection of long- or fast-acting insulin analogs with slightly modified primary sequences compared to human insulin. Assays that are capable of detecting all insulin analogs are desired, not only for medical management of diabetes and severe hypoglycemia but also for sports anti-doping and toxicology.

Volumetric mass spectrometry protein arrays.

Affinity mass spectrometry is a proteomics approach for selectively isolating target proteins from complex biological fluids for mass spectrometric analysis. When executed in high throughput mode through affinity pipets, the resulting volumetric mass spectrometry arrays enable rapid protein assaying from hundreds of samples. Furthermore, in combination with postcapture proteolytic degradation, this top-down proteomics approach can reveal structural features (i.e., modifications) in the protein sequences that are result of posttranslational modifications and/or point mutations. Described here in greater detail are the individual steps of the high throughput combination of affinity protein capture in antibody-derivatized affinity pipets, protein elution, and protein processing through enzyme-derivatized mass spectrometry targets.

Changes in low-density lipoprotein size phenotypes associate with changes in apolipoprotein C-III glycoforms after dietary interventions.

BACKGROUND: The presence of small dense low-density lipoprotein (LDL) is associated with obesity, type II diabetes, and an increased risk for cardiovascular disease. Apolipoprotein C-III (apoC-III) is involved in the formation of small dense LDL, but the exact mechanisms are still not well defined. ApoC-III is a glycosylated apolipoprotein, with 3 major glycoforms: apoC-III0, apoC-III1, and apoC-III2 that contain 0, 1, or 2 molecules of sialic acid, respectively. In our previous work, we reported an association among apoC-III0 and apoC-III1, but not apoC-III2 with fasting plasma triglyceride levels in obesity and type II diabetes. OBJECTIVE: The goal of this study was to determine the relationship between changes in the major apoC-III glycoforms and small dense LDL levels after dietary interventions. METHODS: Mass spectrometric immunoassay was performed on fasting plasma samples from 61 subjects who underwent either a high-carbohydrate diet (n = 34) or a weight loss intervention (n = 27). RESULTS: After both dietary interventions, changes in total apoC-III concentrations were associated with changes in LDL peak particle diameter (r = -0.58, P < .0001). Increases in total apoC-III concentrations after the high-carbohydrate diet were associated with decreases in LDL size (r = -0.53, P = .001), and decreases in apoC-III concentrations after weight loss were associated with increases in LDL peak particle diameter (r = -0.54, P = .004). Changes in concentrations of apoC-III1 and apoC-III0, but not apoC-III2, were associated with changes in LDL peak particle diameter in both the weight loss and high-carbohydrate interventions. CONCLUSIONS: We conclude that apoC-III0 and apoC-III1, but not apoC-III2 are associated with the formation of small dense LDL.

Surface plasmon resonance mass spectrometry for protein analysis.

The combination of surface plasmon pesonance (SPR) and mass spectrometry (MS) provides a unique methodology for studying proteins and their interactions. SPR is utilized to assess protein quantitative variations and the kinetic aspects of protein interactions, whereas MS complements the analysis by providing an exclusive look at the structural features of the interacting proteins via measurement of their mass. Thus, intrinsic protein structural modifications that go unregistered via the SPR detection can readily be assessed from the MS data. The purpose of this chapter is dissemination of the procedures and protocols for successful SPR-MS analysis. The individual steps of the complete SPRMS process are illustrated via analysis of cardiac troponin I (cTnI).

High-throughput affinity mass spectrometry.

Affinity mass spectrometry (AMS) is a proteomics approach for selectively isolating target protein(s) from complex biological fluids for mass spectrometric analysis. The resulting high-content mass spectrometry (MS) data show the unique MS protein signatures (wild-type, posttranslationally modified, as well as genetically modified forms of the protein target) that are present within a biological sample. Information regarding such protein diversity is normally lost in classical proteomic or immunoassay analyses. This chapter presents a step-by-step description of high-throughput AMS in the population proteomic screening of the human plasma protein cystatin C.

Mass spectrometric immunoassays for discovery, screening and quantification of clinically relevant proteoforms.

Human proteins can exist as multiple proteoforms with potential diagnostic or prognostic significance. MS top-down approaches are ideally suited for proteoforms identification because there is no prerequisite for a priori knowledge of the specific proteoform. One such top-down approach, termed mass spectrometric immunoassay utilizes antibody-derivatized microcolumns for rapid and contained proteoforms isolation and detection via MALDI-TOF MS. The mass spectrometric immunoassay can also provide quantitative measurement of the proteoforms through inclusion of an internal reference standard into the analytical sample, serving as normalizer for all sample processing and data acquisition steps. Reviewed here are recent developments and results from the application of mass spectrometric immunoassays for discovery of clinical correlations of specific proteoforms for the protein biomarkers RANTES, retinol binding protein, serum amyloid A and apolipoprotein C-III.

Mass Spectrometric Immunoassays in Characterization of Clinically Significant Proteoforms.

Proteins can exist as multiple proteoforms in vivo, as a result of alternative splicing and single-nucleotide polymorphisms (SNPs), as well as posttranslational processing. To address their clinical significance in a context of diagnostic information, proteoforms require a more in-depth analysis. Mass spectrometric immunoassays (MSIA) have been devised for studying structural diversity in human proteins. MSIA enables protein profiling in a simple and high-throughput manner, by combining the selectivity of targeted immunoassays, with the specificity of mass spectrometric detection. MSIA has been used for qualitative and quantitative analysis of single and multiple proteoforms, distinguishing between normal fluctuations and changes related to clinical conditions. This mini review offers an overview of the development and application of mass spectrometric immunoassays for clinical and population proteomics studies. Provided are examples of some recent developments, and also discussed are the trends and challenges in mass spectrometry-based immunoassays for the next-phase of clinical applications.

Posttranslational modifications of apolipoprotein A-II proteoforms in type 2 diabetes.

BACKGROUND: Apolipoprotein A-II (apoA-II) is the second most abundant protein in high-density lipoprotein particles. However, it exists in plasma in multiple forms. The effect of diabetes on apoA-II proteoforms is not known. OBJECTIVE: Our objective was to characterize plasma apoA-II proteoforms in participants with and without type 2 diabetes. METHODS: Using a novel mass spectrometric immunoassay, the relative abundance of apoA-II proteoforms was examined in plasma of 30 participants with type 2 diabetes and 25 participants without diabetes. RESULTS: Six apoA-II proteoforms (monomer, truncated TQ monomer, truncated Q monomer, dimer, truncated Q dimer, and truncated 2Qs dimer) and their oxidized proteoforms were identified. The ratios of oxidized monomer and all oxidized proteoforms to the native apoA-II were significantly greater in the diabetic group (P = .004 and P = .005, respectively) compared with the nondiabetic group. CONCLUSION: The relative abundance of oxidized apoA-II is significantly increased in type 2 diabetes.