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

Nedelkov, Dobrin; Niederkofler, Eric E.; Oran, Paul E.; Peterman, Scott; Nelson, Randall W.

doi:10.1016/j.jprot.2017.08.001

Cited by 28 publications

(17 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…hormones, immune factors, metabolic regulators) plays a key role in clinical diagnosis, because of the importance of these factors in maintaining homeostasis and their functional disruption in diverse disease states. Typically, analysis of these small protein factors is performed with assays employing antibodies in either ELISAs, or in specific enrichment procedures before targeted LC-MS analysis (1,2). Although these assays can be fast and sensitive, they are expensive and typically only quantify one protein at a time.…”

mentioning

confidence: 99%

Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma

Harney

Hutchison

et al. 2019

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Small proteins in plasma regulate diverse processes from metabolism to immune functions and are difficult to study, because of the large dynamic range of protein abundance. To facilitate their detection, we developed a method using protein dissociation, precipitation and size exclusion chromatography called SPEA. Herein, we demonstrate SPEA is capable of reproducible characterization of known small proteins and identification of novel factors such as C5ORF46. When applied to study humans undergoing intermittent fasting, novel changes in iron regulation were observed.

show abstract

mentioning

confidence: 99%

Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma

Harney

Hutchison

et al. 2019

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…Compared with other LC-MS-based methods for insulin quantification [12], the present approach uses a small sample volume of 250 μL and includes a simple and automated sample preparation, both contributing to its high usability. Thanks to its ease of use and rapid run time (7.5 min per sample or 12 h to run a 96-well plate), the method is highly suitable for studies with a large number of blood samples such as pharmacokinetic studies.…”

Section: Discussionmentioning

confidence: 99%

“…Regardless of their availability, all immunometric assays are unable to separate different insulin variants in a sample, suffer from interferences and have limited linear dynamic ranges [8,10]. Thus, development of mass spectrometry-based methods that show better performance in terms of specificity, selectivity and dynamic range is of high interest [11,12]. In this context, mass spectrometric immunoassays (MSIA) that couple immunopurification with liquid chromatography mass spectrometry (LC-MS) are currently regarded as the most promising approach [12,13].…”

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

“…Thus, development of mass spectrometry-based methods that show better performance in terms of specificity, selectivity and dynamic range is of high interest [11,12]. In this context, mass spectrometric immunoassays (MSIA) that couple immunopurification with liquid chromatography mass spectrometry (LC-MS) are currently regarded as the most promising approach [12,13]. Although quantification of IDeg in human plasma using LC-MS has been recently demonstrated [14], a comprehensive method validation that meets the criteria set forth by the US Food and Drug Administration (FDA), essential for clinical trials, has not yet been performed.…”

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

See 1 more Smart Citation

Rapid quantification of insulin degludec by immunopurification combined with liquid chromatography high-resolution mass spectrometry

et al. 2020

Self Cite

View full text Add to dashboard Cite

Insulin degludec is an ultra-long-acting insulin analogue that is increasingly being used in diabetes due to its favourable efficacy and safety profile. Thus, there is an increasing demand for a reliable and specific analytical method to quantify insulin degludec for research, pharmaceutical industry and clinical applications. We developed and validated an automated, high-throughput method for quantification of insulin degludec in human blood samples across the expected clinical range combining immunopurification with high-resolution mass spectrometry. Validation was performed according to the requirements of the US Food and Drug Administration. The method satisfyingly met the following parameters: lower limit of quantification (120 pM), linearity, accuracy (error < 5%), precision (CV < 7.7%), selectivity, carry-over, recovery (89.7–97.2%), stability and performance in the presence of other insulin analogues. The method was successfully applied to clinical samples of patients treated with insulin degludec showing a good correlation with the administered dose (r2 = 0.78). High usability of the method is supported by the small specimen volume, automated sample processing and short analysis time. In conclusion, this reliable, easy-to-use and specific mass spectrometric insulin degludec assay offers great promise to address the current unmet need for standardized insulin analytics in academic and industrial research.

show abstract

“…Unlike a bottom-up workflow, which is typically more laborious, lengthy, and with sample preparation that can cause erroneous modifications, 36 topdown strategies, [37][38][39] in which mAbs are left intact, can largely overcome these artifacts. [40][41][42][43][44] Additionally, CEX has greater resolving power as compared to RP LC and allows complete separation of intact antibody molecules even with one charged modification or a modification leading to partial net charge change. 7,10,23,29,35 With a few exceptions, 45 RP separation of protein charge variants with one modification is typically relatively poor, 46,47 but RP LC-MS is sensitive and easily amenable to top-down analysis.…”

Section: Introductionmentioning

confidence: 99%

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

Shi

Xiao

Dillon

et al. 2020

mAbs

View full text Add to dashboard Cite

2020) Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis, mAbs, 12:1, 1739825, ABSTRACT Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatographytop-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping. ARTICLE HISTORY

show abstract

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

Cited by 28 publications

References 64 publications

Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma

Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma

Rapid quantification of insulin degludec by immunopurification combined with liquid chromatography high-resolution mass spectrometry

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

Contact Info

Product

Resources

About