Matching Items (11)

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Serum Amyloid A Truncations in Type 2 Diabetes Mellitus

Description

Serum Amyloid A (SAA) is an acute phase protein complex consisting of several abundant isoforms. The N- terminus of SAA is critical to its function in amyloid formation. SAA is

Serum Amyloid A (SAA) is an acute phase protein complex consisting of several abundant isoforms. The N- terminus of SAA is critical to its function in amyloid formation. SAA is frequently truncated, either missing an arginine or an arginine-serine dipeptide, resulting in isoforms that may influence the capacity to form amyloid. However, the relative abundance of truncated SAA in diabetes and chronic kidney disease is not known.
Methods
Using mass spectrometric immunoassay, the abundance of SAA truncations relative to the native variants was examined in plasma of 91 participants with type 2 diabetes and chronic kidney disease and 69 participants without diabetes.
Results
The ratio of SAA 1.1 (missing N-terminal arginine) to native SAA 1.1 was lower in diabetics compared to non-diabetics (p = 0.004), and in males compared to females (p<0.001). This ratio was negatively correlated with glycated hemoglobin (r = −0.32, p<0.001) and triglyceride concentrations (r = −0.37, p<0.001), and positively correlated with HDL cholesterol concentrations (r = 0.32, p<0.001).
Conclusion
The relative abundance of the N-terminal arginine truncation of SAA1.1 is significantly decreased in diabetes and negatively correlates with measures of glycemic and lipid control.

Contributors

Agent

Created

Date Created
  • 2015-01-21

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Parallel Workflow for High-Throughput (>1,000 Samples/Day) Quantitative Analysis of Human Insulin-Like Growth Factor 1 Using Mass Spectrometric Immunoassay

Description

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

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.

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Agent

Created

Date Created
  • 2014-03-24

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Mass Spectrometric Immunoassays in Characterization of Clinically Significant Proteoforms

Description

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

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.

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Agent

Created

Date Created
  • 2016-03-17

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The Simian Barrier Dilemma: Improving Gene Vaccines for the Future

Description

Since its inception in the early 1990s, the concept of gene vaccines, particularly DNA vaccines, has enticed researchers across the board due to its simple design, flexible modification, and overall

Since its inception in the early 1990s, the concept of gene vaccines, particularly DNA vaccines, has enticed researchers across the board due to its simple design, flexible modification, and overall inexpensive cost of manufacturing. However, the past three decades have proven to be less fruitful than anticipated as scientists have yet to tackle the issue of inducing a strong enough response in humans and non-human primates to protect against foreign pathogens, an issue that has since been coined as the “simian barrier.” This appears to be a human/primate barrier as protective vaccines have been produced for other mammals. Despite millions of dollars in research along with some of the world’s brightest minds chipping in to resolve this, there has yet to be any truly viable solution to overcoming this barrier. With current research illustrating effective applications of RNA vaccines in humans, these studies may be uncovering the solution to the largely unsolved simian barrier dilemma. If vaccines using RNA, the transcribed version of DNA, are effective in humans, the problem may be inefficient transcription of the DNA. This may be attributable to a DNA promoter that has insufficient activity in primates. Additionally, with DNA vaccines being even cheaper and easier to manufacture than RNA vaccines, along with having no required cold chain for distribution, this concept remains more promising than RNA vaccines that are further along in clinical trials.

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Agent

Created

Date Created
  • 2020-12

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

Description

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

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.

Contributors

Agent

Created

Date Created
  • 2015-06-15

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A General Method to Discover Epitopes from Sera

Description

Antigen-antibody complexes are central players in an effective immune response. However, finding those interactions relevant to a particular disease state can be arduous. Nonetheless many paths to discovery have been

Antigen-antibody complexes are central players in an effective immune response. However, finding those interactions relevant to a particular disease state can be arduous. Nonetheless many paths to discovery have been explored since deciphering these interactions can greatly facilitate the development of new diagnostics, therapeutics, and vaccines. In silico B cell epitope mapping approaches have been widely pursued, though success has not been consistent. Antibody mixtures in immune sera have been used as handles for biologically relevant antigens, but these and other experimental approaches have proven resource intensive and time consuming. In addition, these methods are often tailored to individual diseases or a specific proteome, rather than providing a universal platform. Most of these methods are not able to identify the specific antibody’s epitopes from unknown antigens, such as un-annotated neo antigens in cancer. Alternatively, a peptide library comprised of sequences unrestricted by naturally-found protein space provides for a universal search for mimotopes of an antibody’s epitope. Here we present the utility of such a non-natural random sequence library of 10,000 peptides physically addressed on a microarray for mimotope discovery without sequence information of the specific antigen. The peptide arrays were probed with serum from an antigen-immunized rabbit, or alternatively probed with serum pre-absorbed with the same immunizing antigen. With this positive and negative screening scheme, we identified the library-peptides as the mimotopes of the antigen. The unique library peptides were successfully used to isolate antigen-specific antibodies from complete immune serum. Sequence analysis of these peptides revealed the epitopes in the immunized antigen. We present this method as an inexpensive, efficient method for identifying mimotopes of any antibody’s targets. These mimotopes should be useful in defining both components of the antigen-antibody complex.

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Agent

Created

Date Created
  • 2016-06-14

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

Description

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

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.

Contributors

Agent

Created

Date Created
  • 2016-03-25

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Delineation of Concentration Ranges and Longitudinal Changes of Human Plasma Protein Variants

Description

Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs) and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo.

Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs) and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo. Tailored strategies are needed to study these protein variants and understand their role in health and disease. In this work we utilized quantitative mass spectrometric immunoassays to determine the protein variants concentration of beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin, in a population of 500 healthy individuals. Additionally, we determined the longitudinal concentration changes for the protein variants from four individuals over a 6 month period. Along with the native forms of the four proteins, 13 posttranslationally modified variants and 7 SNP-derived variants were detected and their concentration determined. Correlations of the variants concentration with geographical origin, gender, and age of the individuals were also examined. This work represents an important step toward building a catalog of protein variants concentrations and examining their longitudinal changes.

Contributors

Agent

Created

Date Created
  • 2014-06-23

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The Association of Human Apolipoprotein C-III Sialylation Proteoforms with Plasma Triglycerides

Description

Introduction
Apolipoprotein C-III (apoC-III) regulates triglyceride (TG) metabolism. In plasma, apoC-III exists in non-sialylated (apoC-III[subscript 0a] without glycosylation and apoC-III[subscript 0b] with glycosylation), monosialylated (apoC-III[subscript 1]) or disialylated (apoC-III[subscript 2])

Introduction
Apolipoprotein C-III (apoC-III) regulates triglyceride (TG) metabolism. In plasma, apoC-III exists in non-sialylated (apoC-III[subscript 0a] without glycosylation and apoC-III[subscript 0b] with glycosylation), monosialylated (apoC-III[subscript 1]) or disialylated (apoC-III[subscript 2]) proteoforms. Our aim was to clarify the relationship between apoC-III sialylation proteoforms with fasting plasma TG concentrations.
Methods
In 204 non-diabetic adolescent participants, the relative abundance of apoC-III plasma proteoforms was measured using mass spectrometric immunoassay.
Results
Compared with the healthy weight subgroup (n = 16), the ratios of apoC-III[subscript 0a], apoC-III[subscript 0b], and apoC-III[subscript 1] to apoC-III[subscript 2] were significantly greater in overweight (n = 33) and obese participants (n = 155). These ratios were positively correlated with BMI z-scores and negatively correlated with measures of insulin sensitivity (S[subscript i]). The relationship of apoC-III[subscript 1] / apoC-III[subscript 2] with S[subscript i] persisted after adjusting for BMI (p = 0.02). Fasting TG was correlated with the ratio of apoC-III[subscript 0a] / apoC-III[subscript 2] (r = 0.47, p<0.001), apoC-III[subscript 0b] / apoC-III[subscript 2] (r = 0.41, p<0.001), apoC-III[subscript 1] / apoC-III[subscript 2] (r = 0.43, p<0.001). By examining apoC-III concentrations, the association of apoC-III proteoforms with TG was driven by apoC-III[subscript 0a] (r = 0.57, p<0.001), apoC-III[subscript 0b] (r = 0.56. p<0.001) and apoC-III[subscript 1] (r = 0.67, p<0.001), but not apoC-III[subscript 2] (r = 0.006, p = 0.9) concentrations, indicating that apoC-III relationship with plasma TG differed in apoC-III[subscript 2] compared with the other proteoforms.
Conclusion
We conclude that apoC-III[subscript 0a], apoC-III[subscript 0b], and apoC-III[subscript 1], but not apoC-III[subscript 2] appear to be under metabolic control and associate with fasting plasma TG. Measurement of apoC-III proteoforms can offer insights into the biology of TG metabolism in obesity.

Contributors

Agent

Created

Date Created
  • 2015-12-03

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

Description

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

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.

Contributors

Agent

Created

Date Created
  • 2014-10-14