Selected Reaction Monitoring Measurements Research Articles

Quantitative targeted screening of proteins associated with lung adenocarcinoma cancer by the method of selected reaction monitoring

In the present study, we applied selected reaction monitoring (SRM) to a group of proteins that were previously reported to be associated with lung cancer (Novikova S.E. et al. (2017) Biomeditsinskaya khimiya, 63, 181-210. [1]). Measurements were performed on 59 plasma samples. These samples included: 23 samples of plasma of patients diagnosed with lung adenocarcinoma (LAC), 11 samples of plasma of patients diagnosed with squamous cell lung carcinoma (SqCC), 25 samples of donors with no previous history of oncological diseases, and one pooled sample from each of the above group. As a result of the SRM measurements 52 proteins were detected at least in one individual plasma sample. Statistical analysis showed that there were two groups confidently differentiated by the concentration value of 8 proteins wherein 5 proteins displayed increased level (P00738, P26639, P21926, P08603, P51149) in LAC group and 3 proteins (P51884, O15162, Q8N2K0) indicated diminishing the concentration level towards the control level. Data on protein concentrations obtained for LAC and SqCC did not distinguish the samples by statistical clustering analysis. These potential biomarkers can be used for further development of methods for early diagnostics of lung cancer.

Selected Reaction Monitoring to Measure Proteins of Interest in Complex Samples: A Practical Guide.

Biology and especially systems biology projects increasingly require the capability to detect and quantify specific sets of proteins across multiple samples, for example the components of a biological pathway through a set of perturbation-response experiments. Targeted proteomics based on selected reaction monitoring (SRM) has emerged as an ideal tool to this purpose, and complements the discovery capabilities of shotgun proteomics methods. SRM experiments rely on the development of specific, quantitative mass spectrometric assays for each protein of interest and their application to the quantification of the protein set in various biological samples. SRM measurements are multiplexed, namely, multiple proteins can be quantified simultaneously, and are characterized by a high reproducibility and a broad dynamic range. We provide here a practical guide to the development of SRM assays targeting a set of proteins of interest and to their application to complex biological samples.

Plasma preparation to measure FDA‐approved protein markers by selected reaction monitoring

BackgroundThe development of commercially available panels for human blood plasma screening via selected reaction monitoring (SRM) offers reliable, cost-efficient and highly-standardized discovery and validation of protein biomarkers. However, protein detection by SRM can be hampered by interfering peptide fragment ions. To estimate the influence of interference on protein detection, we performed different types of sample preparation and implemented SRM measurements for well-characterized protein targets approved by the US Food and Drug Administration.MethodsWe used the PlasmaDeepDive™ SRM assay from BiognoSYS AG for absolute quantification of 18 proteins in 19 samples of human plasma using three different protocols for sample preparation. SRM measurements were performed using iRT standards for retention time normalization and isotopically-labeled reference peptides for absolute quantification. SpectroDive™ software was used for automated detection of reliable peak groups.ResultsFourteen targeted proteins were quantitatively measured in more than half of the samples. Depletion of highly-abundant plasma proteins and peptide fraction clean-up on centrifuge plates resulted in detection of all 18 targeted proteins in femtomolar to picomolar concentrations.ConclusionsIt was shown that commercially designed SRM kits are suitable for SRM detection of well-established plasma/serum biomarkers.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-015-0071-4) contains supplementary material, which is available to authorized users.

Abstract 535: Selected reaction monitoring (SRM)-based assay for the quantification of biomarkers in human breast cancer tissue

Abstract Background: Breast cancer is a heterogeneous disease that has remained the second leading cause of cancer-related death in women despite the advances in research over the past decades. Numerous studies have shown that just like in other cancers, early detection drastically improves survival rates. Breast cancer diagnosis is currently dependent on the differential expression of key proteins (biomarkers) that allow for classification, staging and identification of proper therapies. However, because of its heterogeneity, accurate measurement of these biomarkers remains challenging and subjective. Antibody-based techniques such as immunohistochemistry are currently the standard methods of biomarker measurements in breast cancer. With the improvement of genomic and proteomic technology, the number of validated biomarkers is increasing at a faster pace than the number of corresponding clinical assays, preventing the quick translation from the bench to the clinic. Selected reaction monitoring (SRM) is a robust and accurate mass spectrometry-based method that can be used to simultaneously quantify subgroups of protein-specific peptides within any complex protein mixture. The goal of this study is to develop an SRM-based assay for simultaneous detection of 40 biomarkers in tissues. Method: Our panel of biomarkers consists of well-established proteins whose expression correlates with breast cancer aggressiveness and therapy, such as estrogen receptor (ER), HER2 and progesterone receptor (PR). It also includes cellular characterization markers like E-cadherin, fibronectin and vimentin that can aid in better categorization of cancer types. Peptides specific to beta-actin and GAPDH are used as internal controls. We have access to over 2000 characterized breast tissue samples available at the UT Southwestern Medical Center tissue repository. These paraffin-embedded tissues are laser-capture micro-dissected and digested prior to SRM measurements. For validation, we compare the SRM measurements to current bench-top techniques such as immunohistochemistry and Western blot. Results: Using our current method, we have successfully quantified the levels of ER, PR, Her2, and fibronectin on 2.4mm2 breast tissues. Additional experiments are underway to quantify the levels of these and additional proteins in additional samples and to compare their expression to their clinical and pathology reports. Conclusion: Development of high quality antibody assays is time consuming and requires extensive resources and efforts. In addition immunohistochemistry assays cannot be multiplexed. This project will provide a faster, cost-effective, and multiplexable method for breast cancer biomarkers detection and quantification. Future studies will focus on measuring the levels of the same biomarkers in serum samples. Altogether, it will allow for a quick validation platform as new biomarkers are identified. Citation Format: Alimatou M. Tchafa, Andrew S. Lemoff, Hamid Mirzaei. Selected reaction monitoring (SRM)-based assay for the quantification of biomarkers in human breast cancer tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 535. doi:10.1158/1538-7445.AM2015-535

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

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 native forms of challenging protein targets.

Considerations on selected reaction monitoring experiments: Implications for the selectivity and accuracy of measurements

Targeted MS analyses based on selected reaction monitoring (SRM) has enabled significant achievements in proteomic quantification, such that its application to clinical studies has augured great advancements for life sciences. The approach has been challenged by the complexity of clinical samples that affects the selectivity of measurements, in many cases limiting analytical performances to a larger extent than expected. This Personal Perspective discusses some insight to better comprehend the mismatch between the often underestimated sample complexity and the selectivity of SRM measurements performed on a triple quadrupole instrument. The implications for the design and evaluation of SRM assays are discussed and illustrated with selected examples, providing a baseline for a more critical use of the technique in the context of clinical samples and to evaluate alternative methods.

18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.

MProphet: automated data processing and statistical validation for large-scale SRM experiments

Selected reaction monitoring (SRM) is a targeted mass spectrometric method that is increasingly used in proteomics for the detection and quantification of sets of preselected proteins at high sensitivity, reproducibility and accuracy. Currently, data from SRM measurements are mostly evaluated subjectively by manual inspection on the basis of ad hoc criteria, precluding the consistent analysis of different data sets and an objective assessment of their error rates. Here we present mProphet, a fully automated system that computes accurate error rates for the identification of targeted peptides in SRM data sets and maximizes specificity and sensitivity by combining relevant features in the data into a statistical model.