Selected reaction monitoring, also known as multiple reaction monitoring, is a powerful targeted mass spectrometry approach for a confident quantitation of proteins/peptides in complex biological samples. In recent years, its optimization and application have become pivotal and of great interest in clinical research to derive useful outcomes for patient care. Thus, selected reaction monitoring/multiple reaction monitoring is now used as a highly sensitive and selective method for the evaluation of protein abundances and biomarker verification with potential applications in medical screening. This review describes technical aspects for the development of a robust multiplex assay and discussing its recent applications in cardiovascular proteomics: verification of promising disease candidates to select only the highest quality peptides/proteins for a preclinical validation, as well as quantitation of protein isoforms and post-translational modifications. 相似文献
Introduction: Post-translational modifications (PTMs) have an important role in the regulation of protein function, localization, and interaction with other molecules. PTMs apply a dynamic control of proteins in both physiological and pathological conditions. The study of disease-specific PTMs allows identifying potential biomarkers and developing effective drugs. Enrichment techniques combined with high-resolution mass spectrometry (MS)/MS analysis provide attractive results on PTM characterization. Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) is a powerful targeted assay for the quantitation and validation of PTMs in complex biological samples.
Areas covered: The most frequent PTMs are described in terms of biological role and analytical methods commonly used to detect them. The applications of SRM/MRM for the absolute quantitation of PTMs are reported, and a specific section is focused on PTM detection in proteins that are involved in the cardiovascular system and heart diseases.
Expert commentary: PTM characterization in relation to disease pathology is still in progress, but targeted proteomics by LC-MS/MS has significantly upgraded our knowledge in the last few years. Advances in enrichment strategies and software tools will facilitate the interpretation of high PTM complexity. Promising studies confirm the great potential of SRM/MRM to study PTMs in the cardiovascular field, and PTMomics could be very useful in the clinical perspective. 相似文献
Mass spectrometry-based plasma proteomics is a field where intense research has been performed during the last decade. Being closely linked to biomarker discovery, the field has received a fair amount of criticism, mostly due to the low number of novel biomarkers reaching the clinic. However, plasma proteomics is under gradual development with improvements on fractionation methods, mass spectrometry instrumentation and analytical approaches. These recent developments have contributed to the revival of plasma proteomics. The goal of this review is to summarize these advances, focusing in particular on fractionation methods, both for targeted and global mass spectrometry-based plasma analysis. 相似文献
The advances in high-resolution mass spectrometry instrumentation, capable of accurate mass measurement and fast acquisition, have enabled new approaches for targeted quantitative proteomics. More specifically, analyses performed on quadrupole-orbitrap mass spectrometers operated in parallel reaction monitoring (PRM) mode leverage the intrinsic high resolving power and trapping capabilities. The PRM technique offers unmatched degrees of selectivity and analytical sensitivity, typically required to analyze peptides in complex samples, such as those encountered in biomedical research or clinical studies. The features of PRM have provoked a paradigm change in targeted experiments, by decoupling acquisition and data processing. It has resulted in a new analytical workflow comprising distinct methods for each step, thus enabling much larger flexibility. The PRM technique was further enhanced by a new data acquisition scheme, allowing dynamic parameter settings. The potential of the technique may radically impact future quantitative proteomics studies. 相似文献
A crucial part of a successful systems biology experiment is an assay that provides reliable, quantitative measurements for each of the components in the system being studied. For proteomics to be a key part of such studies, it must deliver accurate quantification of all the components in the system for each tested perturbation without any gaps in the data. This will require a new approach to proteomics that is based on emerging targeted quantitative mass spectrometry techniques. The PeptideAtlas Project comprises a growing, publicly accessible database of peptides identified in many tandem mass spectrometry proteomics studies and software tools that allow the building of PeptideAtlas, as well as its use by the research community. Here, we describe the PeptideAtlas Project, its contents and components, and show how together they provide a unique platform to select and validate mass spectrometry targets, thereby allowing the next revolution in proteomics. 相似文献
Today, proteomics usually compares clinical samples by use of bottom-up profiling with high resolution mass spectrometry, where all protein products of a single gene are considered as an integral whole. At the same time, proteomics of proteoforms, which considers the variety of protein species, offers the potential to discover valuable biomarkers. Proteoforms are protein species that arise as a consequence of genetic polymorphisms, alternative splicing, post-translational modifications and other less-explored molecular events. The comprehensive observation of proteoforms has been an exclusive privilege of top-down proteomics. Here, we review the possibilities of a bottom-up approach to address the microheterogeneity of the human proteome. Special focus is given to shotgun proteomics and structure-based bioinformatics as a source of hypothetical proteoforms, which can potentially be verified by targeted mass spectrometry to determine the relevance of proteoforms to diseases. 相似文献
The development of mass spectrometry (MS) technologies has brought the ability to gather massive amounts of data characterising the proteomes of complex mixtures. A major focus in proteomics is to leverage this data-gathering capability to conduct comparative analyses of biofluids from healthy and disease-affected patients for the identification of highly specific biomarkers and/or the development of MS-based diagnostic platforms. Much effort has gone into optimising the biofluid proteome coverage that can be obtained using these technologies, leaving proteomics poised to make an important impact in disease diagnostics in the future. 相似文献
d-Glucose dehydrogenase [β-d-glucosc: NAD(P) oxidoreductase (EC 1.1.1.47)] was synthesized derepressively in a mutant of a Bacillus species which was isolated as an improved strain for d-ribose production. The enzyme was very unstable and inactivated during storage or column chromatography. The inactivation was prevented in the presence of NAD+, NADP+ or certain salts. The inactive enzyme was reactivated by the addition of NAD+, NADH, NADP+, NADPH, AMP, ADP, ATP or certain salts. The molecular weights of the inactive and active form of the enzyme were estimated to be about 45,000 and 80,000, respectively, by Sephadex G–150 gel filtration. Thus, it seems that the enzyme activity is regulated by monomer-dimer interconversion of the enzyme molecule. 相似文献
Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently‐used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower‐abundance proteins in Arabidopsis. NPAS was combined with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox ( http://suba.live ). 相似文献
Mass spectrometry (MS) -based proteomics has become an indispensable tool with broad applications in systems biology and biomedical research. With recent advances in liquid chromatography (LC) and MS instrumentation, LC–MS is making increasingly significant contributions to clinical applications, especially in the area of cancer biomarker discovery and verification. To overcome challenges associated with analyses of clinical samples (for example, a wide dynamic range of protein concentrations in bodily fluids and the need to perform high throughput and accurate quantification of candidate biomarker proteins), significant efforts have been devoted to improve the overall performance of LC–MS-based clinical proteomics platforms. Reviewed here are the recent advances in LC–MS and its applications in cancer biomarker discovery and quantification, along with the potentials, limitations and future perspectives. 相似文献
Insulin resistance (IR) underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of IR, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in IR in different WAT depots by developing a targeted proteomics approach to quantitatively compare the abundance of 42 nuclear proteins in subcutaneous and visceral WAT from a commonly used insulin-resistant mouse model, Lepr(db/db), and from C57BL/6J control mice. The most differentially expressed proteins were important in adipogenesis, as confirmed by siRNA-mediated depletion experiments, suggesting a defect in adipogenesis in visceral, but not subcutaneous, insulin-resistant WAT. Furthermore, differentiation of visceral, but not subcutaneous, insulin-resistant stromal vascular cells (SVCs) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVCs to preadipocyte cell culture models made insulin resistant by different stimuli. The insulin-resistant visceral SVC protein abundance profile correlated most with preadipocyte cell culture cells treated with both palmitate and TNFα. Together, our study introduces a method to simultaneously measure and quantitatively compare nuclear protein expression patterns in primary adipose tissue and adipocyte cell cultures, which we show can reveal relationships between differentiation and disease states of different adipocyte tissue types. 相似文献
So far, mass spectrometry-based targeted proteomics is the most sensitive approach to answer and address specific biological questions in an accurate and quantitative fashion. However, the data analysis design used for such quantification varies in the field leading to discrepancies in the reported values. In this study, different quantification strategies based on calibration curves were evaluated and compared. The best accuracy and coefficient of variation was achieved by ratio to ratio calibration curves. We applied the ratio to ratio quantification approach to analyze very low abundant insulin signaling proteins such as PIK3RA (0.10–0.93 fmol/μg), AKT1 (0.1–0.39 fmol/μg), and the insulin receptor (0.22–2.62 fmol/μg) in a fat cell model and demonstrated the adaptation of this pathway at different states of insulin sensitivity. 相似文献