Dobrin Nedelkov

Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum

OBJECTIVES The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimers, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.

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)

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC‐MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi‐automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.

Targeted Selected Reaction Monitoring Mass Spectrometric Immunoassay for Insulin-like Growth Factor 1

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.

New and emerging proteomic techniques

On-Chip Protein Synthesis for Making Microarrays Niroshan Ramachandran, Eugenie Hainsworth, Gokhan Demirkan, and Joshua LaBaer RCA-Enhanced Protein Detection Arrays Brian B. Haab and Paul M. Lizardi Antibody Microarrays Using Resonance Light-Scattering Particles for Detection Bernhard H. Geierstanger, Petri Saviranta, and Achim Brinker Chemical Proteomics Profiling of Proteasome Activity Martijn Verdoes, Celia R. Berkers, Bogdan I. Florea, Paul F. van Swieten, Herman S. Overkleeft, and Huib Ovaa Two-Dimensional Difference Gel Electrophoresis Terence L. Wu Oligomeric States of Proteins Determined by Size-Exclusion Chromatography Coupled With Light Scattering, Absorbance, and Refractive Index Detectors Ewa Folta-Stogniew Surface Plasmon Resonance Imaging Measurements of Protein Interactions With Biopolymer Microarrays Terry T. Goodrich, Alastair W. Wark, Robert M. Corn, and Hye Jin Lee Surface Plasmon Resonance Mass Spectrometry for Protein Analysis Dobrin Nedelkov and Randall W. Nelson High-Throughput Affinity Mass Spectrometry Urban A. Kiernan, Dobrin Nedelkov, Eric E. Niederkofler, Kemmons A. Tubbs, and Randall W. Nelson Isotope-Coded Affinity Tags for Protein Quantification Christopher M. Colangelo and Kenneth R. Williams Proteomic Analysis by Multidimensional Protein Identification Technology Laurence Florens and Michael P. Washburn Isolation of Glycoproteins and Identification of Their N-Linked Glycosylation Sites Hui Zhang and Ruedi Aebersold N-Glycosylation Analysis Using the StrOligo Algorithm Martin Ethier, Daniel Figeys, and Helene Perreault MALDI-MS Data Analysis for Disease Biomarker Discovery Weichuan Yu, Baolin Wu, Junfeng Liu, Xiaoye Li, Kathy Stone, Kenneth R. Williams, and Hongyu Zhao Using the Global Proteome Machine for Protein Identification Ronald C.Beavis Index

Abstract 2508: Mass spectrometric immunoassay for insulin-like growth factor 1.

Mass Spectrometric Immunoassays (MSIA) are protein quantification methods that utilize mass spectrometric detection for quantification of targeted protein analytes affinity retrieved by surface-immobilized antibodies. This rather straightforward concept is realized through affinity pipette devices (MSIA-Tips) that enable high-throughput assaying of hundreds of samples per day. Described here is the development and validation of mass spectrometric immunoassay for insulin-like growth factor 1 (IGF1). MSIA-Tips derivatized with anti-IGF-1 antibody were used for affinity isolation of IGF1 from human samples. Long R3 (LR3) IGF1, spiked into samples prior to the affinity isolation, served as internal reference standard for signal normalization and IGF1 quantification. Following capture and elution, both the intact proteins and their surrogate tryptic peptides were analyzed via LC/MS. Linear standard curves were built that spanned the range of 1 to 1,500 ng/mL. The assay exhibited intra- and inter-assay precisions of Citation Format: Dobrin Nedelkov, Eric Niederkofler, David Phillips, Bryan Krastins, Urban Kiernan, Kemmons Tubbs, Mary Lopez. Mass spectrometric immunoassay for insulin-like growth factor 1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2508. doi:10.1158/1538-7445.AM2013-2508

Proteomics of Human Urine

Urine is one of the most interesting and useful human body fluids for clinical proteomics studies because of its availability in almost all patients and ease of collection without any invasive or painful procedure. Proteomic analysis of human urine offers invaluable information not only for a better understanding of the normal renal physiology and pathophysiology of kidney/kidney-related diseases, but also for biomarker discovery. This chapter provides principles, brief methods, and applications of various proteomic technologies (i.e., gel-based method, liquid chromatographybased approach, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, capillary electrophoresis coupled to mass spectrometry, and mass spectrometric immunoassay) to examine the human urine. At the end, perspectives and future directions in the field of urinary proteomics are discussed.