SRM and Other MS-based Quantification of Proteins

HomeResearch TopicsSRM-based and Other Targeted Methods for Protein QuantificationSRM and Other MS-based Quantification of Proteins
SRM and Other MS-based Quantification of Proteins 2016-11-10T20:21:00+00:00

Notable peer-reviewed articles–using selected reaction monitoring (SRM) mass spectrometry for quantification of proteins in complex biological mixtures.

 

Articles of note

Figure 1Plasma proteome profiling to assess human health and disease (free full text)

Blood, plasma, and serum are the predominant samples used for diagnostic analyses in clinical practice and are available in biobanks from thousands of clinical studies (Végvári et al., 2011). The quantitative analysis of individual plasma proteins by immunoassays is used in daily clinical diagnostics. However, immunoassays have inherent limitations with regard to multiplexing, their specificity for protein isoforms, and their incompatibility with hypothesis-free investigations. Mass spectrometry (MS)-based proteomics is a technology that could address all of these limitations and that should be capable of discovering biomarkers in this easily accessible body fluid (Anderson, 2014). Read more ›

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics (free full text)

Selected reaction monitoring (SRM) – also known as multiple reaction monitoring (MRM) – has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for, e.g. detecting low-abundance biomarkers likely present at the low ng/mL to pg/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in cells or tissues. Read more ›

Analytical strategies for the global quantification of intact proteins

The quantification of intact proteins is a relatively recent development in proteomics. In eukaryotic organisms, proteins are present as multiple isoforms as the result of variations in genetic code, alternative splicing, post-translational modification and other processing events. Understanding the identities and biological functions of these isoforms and how their concentrations vary across different states is the central goal of proteomics. To date, the bulk of proteomics research utilizes a “bottom-up” approach, digesting proteins into their more manageable constitutive peptides, but sacrificing information about the specific isoform and combinations of post-translational modifications present on the protein. Read more ›

Automated selected reaction monitoring data analysis workflow for large-scale targeted proteomic studies

Targeted proteomics based on selected reaction monitoring (SRM) mass spectrometry is commonly used for accurate and reproducible quantification of protein analytes in complex biological mixtures. Strictly hypothesis-driven, SRM assays quantify each targeted protein by collecting measurements on its peptide fragment ions, called transitions. To achieve sensitive and accurate quantitative results, experimental design and data analysis must consistently account for the variability of the quantified transitions. This consistency is especially important in large experiments, which increasingly require profiling up to hundreds of proteins over hundreds of samples. Read more ›

Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins

Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. Read more ›

Multiplexed MS/MS for improved data-independent acquisition (free full text)

In mass spectrometry-based proteomics, data-independent acquisition (DIA) strategies can acquire a single data set useful for both identification and quantification of detectable peptides in a complex mixture. However, DIA data are noisy owing to a typical five- to tenfold reduction in precursor selectivity compared to data obtained with data-dependent acquisition or selected reaction monitoring. We demonstrate a multiplexing strategy, MSX, for DIA analysis that increases precursor selectivity fivefold. Read more ›

Platform for establishing interlaboratory reproducibility of selected reaction monitoring-based mass spectrometry peptide assays

Mass spectrometry (MS) is an attractive alternative to quantification of proteins by immunoassays, particularly for protein biomarkers of clinical relevance. Reliable quantification requires that the MS-based assays are robust, selective, and reproducible. Thus, the development of standardized protocols is essential to introduce MS into clinical research laboratories. The aim of this study was to establish a complete workflow for assessing the transferability and reproducibility of selected reaction monitoring (SRM) assays between clinical research laboratories. Read more ›

Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins (free full text)

Quantitative LC-MS/MS assays were designed for tryptic peptides representing 53 high and medium abundance proteins in human plasma using a multiplexed multiple reaction monitoring (MRM) approach. Of these, 47 produced acceptable quantitative data, demonstrating within-run coefficients of variation (CVs) (n = 10) of 2-22% (78% of assays had CV <10%). A number of peptides gave CVs in the range 2-7% in five experiments (10 replicate runs each) continuously measuring 137 MRMs, demonstrating the precision achievable in complex digests. Depletion of six high abundance proteins by immunosubtraction significantly improved CVs compared with whole plasma, but analytes could be detected in both sample types. Read more ›

Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions

Selected reaction monitoring (SRM) is a targeted mass spectrometry technique that is emerging in the field of proteomics as a complement to untargeted shotgun methods. SRM is particularly useful when predetermined sets of proteins, such as those constituting cellular networks or sets of candidate biomarkers, need to be measured across multiple samples in a consistent, reproducible and quantitatively precise manner. Here we describe how SRM is applied in proteomics, review recent advances, present selected applications and provide a perspective on the future of this powerful technology. Read more ›

 

Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach (free full text)

Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Read more ›

Targeted quantitation of proteins by mass spectrometry (free full text)

Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Read more ›