Mary F Lopez (Presenter)
Nuclea Biotechnologies
Bio: Dr. Lopez has over 25 years of experience in the fields of Proteomics and Mass Spectrometry and joined Nuclea Biotechnologies in 2015. She received her PhD from the University of Massachusetts Amherst and did her postdoctoral work at MIT where she received an NIH fellowship. Dr Lopez has held leadership positions in several Health related and Biotechnology companies including Millipore Corp., Perkin Elmer and most recently, Thermo Fisher Scientific. She has authored over 70 peer reviewed publications in her field, has been awarded several patents and serves on the Scientific Advisory Board of the Clinical Proteomics Research Center at Massachusetts General Hospital. Dr Lopez is also on the Board of the US Human Proteome Organization (HUPO) and is an associate editor of Clinical Proteomics.
Authorship: Bryan Krastins (1), Gregory Byram (1), Gouri Vadali (1), Maryann Vogelsang (1), Denis Faubert (2), Marie-Soleil Gauthier (2), Omid Hekmat (2), Benoit Coulombe (2), Remi Rabasa-Lhoret (2), Pierre Garne
(1) Nuclea Biotechnologies, Cambridge, MA (2) IRCM (Institute de Recherches Cliniques de Montréal) Montréal, CA, (3) Hopital du Sacre-Coeur de Montreal, (4) Joslin Diabetes Center, Boston, MA
| Short Abstract In the past, the clinical utility of monitoring insulin and proinsulin levels in blood has been low due to the variability between available assays (typically ELISA and Western Blot) and lack of standardization (1-3). The establishment of an accurate and standardized approach would have clinical value in the assessment of endogenous and synthetic analogs of insulin as well as proinsulin and C-peptide. To address this shortcoming, we have developed and validated a multiplexed assay for intact proinsulin, insulin, C-Peptide and synthetic insulin analogs using semi-automated immunoenrichment coupled to high-resolution mass spectrometric detection. The multiplexed assay is rapid (ca 10 mins), precise and sensitive across the complete clinical range for all analytes. |
Long Abstract
Approximately 387 Million people worldwide are living with Diabetes (http://www.idf.org/worlddiabetesday/toolkit/gp/facts-figures ). This number is expected to increase by 205 million by 2035. Currently, the only reliable tests for diagnosing and managing Diabetes are measuring the levels of Hemoglobin A1c and fasting glucose levels in blood.
Accurate measurement of insulin, C-peptide and proinsulin can be useful to identify insulin resistance, hypoglycecemia and pancreatic diseases as well as a host of other pathologies. In addition, an accurate test is useful for measurement of the levels of synthetic versus endogenous insulins in patients undergoing therapy. Other applications of insulin assays include sports doping and forensic testing.
Unfortunately, commercial insulin tests, primarily ELISA-based tests, suffer from a significant degree of cross-reactivity and interference making their clinical usefulness limited (1-3).
Based on a previous study (4), we have developed and validated a multiplexed test that combines immunoenrichment with mass spectrometric detection to quantify endogenous insulin, proinsulin, C-peptide and any known variants present in clinical serum or plasma samples. Our assay is sensitive enough to detect and robustly quantify targeted analytes at sub ng/ml levels while maintaining a wide dynamic range (3 orders of magnitude). The high selectivity of the assay allows confident differentiation of target analyte mass spec signal from the background matrix. The resulting method is universally applicable for precise quantification of proinsulin, endogenous insulin, C-peptide and synthetic insulin variants individually or simultaneously without the need to drastically change the components of the assay.
A crucial advantage of the assay is related to the efficient, selective and simultaneous extraction and enrichment of insulin/proinsulin/C-peptide variants from the plasma/serum matrix. Incorporation of insulin MSIA™ Disposable Automated Research Tips (D.A.R.T.’S, Thermo Scientific™), (as well as custom devices) which integrate affinity micro-columns coated with antibodies into a functional pipette tip, facilitates automated extraction of proinsulin, insulin, C-peptide and insulin variants from serum or plasma through the use of an automated liquid handler. Using this approach, all insulin variants are extracted simultaneously conserving time, buffers and 96 well plates which directly feed into the standardized liquid chromatography mass spectrometry (LC-MS) analysis. Utilizing a pan-insulin Ab allowed incorporation of an internal standard in order to quantify targeted insulin variants as well as to enable quality control/system suitability determination. MSIA enrichment significantly reduces contaminants in the biological matrix, thereby extending the detection and quantitation range for the insulin analysis.
Performing the LC-MS experiments using the Q Exactive mass spectrometer (Thermo Scientific™) facilitates high resolution/accurate mass analysis of the targeted analytes. Detection using HR/AM MS strategies efficiently separate the proinsulin, insulin variant and C-Peptide ion signals from the background matrix. Automated data processing using Pinpoint software (Thermo Scientific™) was performed to determine presence/absence and area under the curve (AUC) values to determine relative/absolute amounts of all targeted analytes. A single workbook within Pinpoint contained all of the targeted insulin variants and corresponding m/z values representing precursor and product ions. Depleting the background matrix with MSIA extraction allowed data extraction using ±5 ppm tolerance. The top six isotopic m/z values per charge state were used for determination of presence/absence, verification, and quantification. A total of 18 extracted ion chromatograms (XICs) per analyte were used for processing. We utilized a synthetic heavy insulin as an internal standard and spiked it into each sample prior to MSIA extraction. Verification was based on isotopic distribution overlap between the relative AUC values per isotope as compared to the theoretical isotopic distribution. A calculated correlation coefficient 0.95 was used as the acceptance criterion for all analytes.
A clear benefit of our method is that the entire validation protocol was simultaneously applied to proinsulin, insulin, C-peptide and all known variants. The validation protocol included the following experiments: calibration curve, spike/recovery, inter/intra day reproducibility, and linearity. Each experiment type was prepared with all samples spiked into the insulin depleted plasma matrix. The calibration curve experiment included a nine-point curve plus neat serum standard and was executed a total of five separate times from the same starting pool. The linearity experiment was conducted five separate times by blending high and low spiked serum, beginning at 100% low and ranging to 100% high with increments of 10%. The spike/recovery experiment included a seven-point calibration curve in full triplicate with neat, low, medium and high serum samples run a total of five times each. The intra day experiment included a seven-point calibration curve in triplicate with low, medium and high serum samples each run with a total of 41 replicates. The inter-day experiment included a seven-point calibration curve in triplicate with low, medium and high serum samples each run with a total of 5 replicates. This process was repeated a total of 20 separate times.
In summary, we have developed and validated a very robust multiplexed mass spec-based assay for the rapid (ca 10 min) quantification of proinsulin, insulin and C-peptide. Our assay can simultaneously and reliably quantify endogenous and synthetic forms of insulin as well as known variants. The development and availability of this assay addresses an urgent clinical need for monitoring the large and rapidly growing population of individuals with Diabetes, obesity and metabolic syndrome.
References & Acknowledgements:
1. Dayaldasani A, Rodríguez Espinosa M, Ocón Sánchez P, Pérez Valero V. Cross-reactivity of insulin analogues with three insulin assays. Ann Clin Biochem. 2015 May;52(Pt 3):312-8. doi: 10.1177/0004563214551613. Epub 2014 Aug 29.
2. Couchman L, Taylor DR, Moniz CF. Analysis of insulin and insulin analogues by mass spectrometry. Ann Clin Biochem OnlineFirst August 3, 2015 doi:10.1177/0004563215597011
3. Parfitt C, Church D, Armston A, Couchman L, Evans C, Wark G, McDonald TJ. Commercial insulin immunoassays fail to detect commonly prescribed insulin analogues. Clin Biochem. 2015 Jul 11. pii: S0009-9120(15)00278-7. doi: 10.1016/j.clinbiochem.2015.07.017.
4. Peterman S, Niederkofler EE, Phillips DA, Krastins B, Kiernan UA, Tubbs KA, Nedelkov D, Prakash A, Vogelsang MS, Schoeder T, Couchman L, Taylor DR, Moniz CF, Vadali G, Byram G, Lopez MF. An automated, high-throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA-HR/AM). Proteomics. 2014 Jun;14(12):1445-56. doi: 10.1002/pmic.201300300.
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