Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers

Monoclonal antibodies that bind the native conformation of proteins are indispensable reagents for the development of immunoassays, production of therapeutic antibodies and delineating protein interaction networks by affinity purification-mass spectrometry. Antibodies generated against short peptides, protein fragments, or even full length recombinant proteins may not bind the native protein form in biological fluids, thus limiting their utility. Here, we report the application of immunocapture coupled with selected reaction monitoring measurements (immunocapture-SRM), in the rapid screening of hybridoma culture supernatants for monoclonal antibodies that bind the native protein conformation. We produced mouse monoclonal antibodies, which detect in human serum or seminal plasma the native form of the human testis-expressed sequence 101 (TEX101) protein-a recently proposed biomarker of male infertility. Pairing of two monoclonal antibodies against unique TEX101 epitopes led to the development of an ELISA for the measurement of TEX101 in seminal plasma (limit of detection: 20 pg/ml) and serum (limit of detection: 40 pg/ml). Measurements of matched seminal plasma samples, obtained from men pre-and post-vasectomy, confirmed the absolute diagnostic specificity and sensitivity of TEX101 for noninvasive identification of physical obstructions in the male reproductive tract. Measurement of male and female serum samples revealed undetectable levels of TEX101 in the systemic circulation of healthy individuals. Immunocapture-SRM screening may facilitate development of monoclonal antibodies and immunoassays against na-

Section: Discussionmentioning

confidence: 99%

Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids*

Korbakis

Brinc

Schiza

et al. 2015

“…Proteotypic tryptic peptides containing isotopically coded amino acids ([ 13 MS is capable of sensitive and accurate protein quantitation based on the quantitation of proteolytic peptides as surrogates for the corresponding intact proteins. Over the past 10 years, MS-based protein quantitation based on the analysis of peptides (in other words, based on "bottom-up" proteomics) has had a profound impact on how biological problems can be addressed (1,2). Although advances in MS instrumentation have contributed to the improvement of MS-based protein quantitation, the use of stable isotopes in quantitative work flows has arguably had the greatest impact in improving the quality and reproducibility of MS-based protein quantitation (3)(4)(5).…”

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confidence: 99%

“…Multiple reaction monitoring (MRM) 1 is a tandem MS (MS/MS) scan mode unique to triple quadrupole MS instrumentation that is capable of rapid, sensitive, and specific quantitation of analytes in highly complex sample matrices (7). MRM is a targeted approach that requires knowledge of the molecular weight of an analyte and its fragmentation behavior under CID.…”

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confidence: 99%

Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma

Kuzyk

Smith

Yang

et al. 2009

482

560

Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ( MS is capable of sensitive and accurate protein quantitation based on the quantitation of proteolytic peptides as surrogates for the corresponding intact proteins. Over the past 10 years, MS-based protein quantitation based on the analysis of peptides (in other words, based on "bottom-up" proteomics) has had a profound impact on how biological problems can be addressed (1, 2). Although advances in MS instrumentation have contributed to the improvement of MS-based protein quantitation, the use of stable isotopes in quantitative work flows has arguably had the greatest impact in improving the quality and reproducibility of MS-based protein quantitation (3-5).The ongoing development of untargeted MS-based quantitation work flows has focused on increasingly exhaustive sample prefractionation methods, at both the protein and peptide levels, with the goal of detecting and quantifying entire proteomes (6). Although untargeted MS-based quantitation work flows have their utility, they are costly in terms of lengthy MS data acquisition and analysis times, and as a result, they are often limited to quantifying differences between small sample sets (n Ͻ 10). To facilitate rapid quantitation of larger, clinically relevant sample sets (n Ͼ 100) there is a need to both simplify sample preparation and reduce MS analysis time.Multiple reaction monitoring (MRM) 1 is a tandem MS (MS/MS) scan mode unique to triple quadrupole MS instrumentation that is capable of rapid, sensitive, and specific quantitation of analytes in highly complex sample matrices (7). MRM is a targeted approach that requires knowledge of the molecular weight of an 1 The abbreviations used are: MRM, multiple reaction monitoring; CE, collision energy; CV, coefficient of variation; CZE, capillary zone electrophoresis; DP, declustering potential; LOQ, limit of quantitation; Q1, quadrupole 1; Q3, quadrupole 3; SIS, stable isotope-labeled standard; XIC, extracted ion chromatogram; iTRAQ, isobaric tags for relative and absolute quantitation. Research

“…However, for many of these newly identified potential biomarkers there are no ELISA kits available, and development of an ELISA kit is time-consuming (6), making it impractical to examine large panels of biomarkers. Although ELISA remains the "gold standard" for clinical applications, mass spectrometry-based quantification analysis has been shown as a possible intermediate technology in the validation process to prioritize candidates for ELISA development (7)(8)(9)(10)(11)(12).…”

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confidence: 99%

Use of an Immunoaffinity-Mass Spectrometry-based Approach for the Quantification of Protein Biomarkers from Serum Samples of Lung Cancer Patients

Nicol¹,

Han²,

Kim³

et al. 2008

106

It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometrybased approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4 -500 ng/ml), tissue factor pathway inhibitor (TFPI) (42-1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2-250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430 -1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinitymultiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins Biomarkers have attracted significant attention for disease diagnosis, prognosis, and therapeutic response monitoring in recent years (1). The development of clinical biomarkers involves discovery, verification, and validation. Progress in genomics and proteomics has led to a large number of potential biomarkers (2-4). However, the promise of these discoveries requires proper verification and validation in the disease of interest (5). The ELISA has been the established platform for quantification of proteins in sera. However, for many of these newly identified potential biomarkers there are no ELISA kits available, and development of an ELISA kit is time-consuming (6), making it impractical to examine large panels of biomarkers. Although ELISA remains the "gold standard" for clinical applications, mass spectrometry-based quantification analysis has been shown as a possible intermediate technology in the validation process to prioritize candidates for ELIS...