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      Mass spectrometric protein maps for biomarker discovery and clinical research

      Expert Review of Molecular Diagnostics
      Informa UK Ltd.
      biomarker discovery, diagnostics, digital maps, mass spectrometry, proteomics, reference maps, selected reaction monitoring, swath ms

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          Abstract

          Among the wide range of proteomic technologies, targeted mass spectrometry (MS) has shown great potential for biomarker studies. To extend the degree of multiplexing achieved by selected reaction monitoring (SRM), we recently developed SWATH MS. SWATH MS is a variant of the emerging class of data-independent acquisition (DIA) methods and essentially converts the molecules in a physical sample into perpetually re-usable digital maps. The thus generated SWATH maps are then mined using a targeted data extraction strategy, allowing us to profile disease-related proteomes at a high degree of reproducibility. The successful application of both SRM and SWATH MS requires the a priori generation of reference spectral maps that provide coordinates for quantification. Herein, we demonstrate that the application of the mass spectrometric reference maps and the acquisition of personalized SWATH maps hold a particular promise for accelerating the current process of biomarker discovery.

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          Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

          Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.
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            The path to personalized medicine.

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              Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics.

              Selected reaction monitoring on a triple quadrupole mass spectrometer is currently experiencing a renaissance within the proteomics community for its, as yet, unparalleled ability to characterize and quantify a set of proteins reproducibly, completely, and with high sensitivity. Given the immense benefit that high resolution and accurate mass instruments have brought to the discovery proteomics field, we wondered if highly accurate mass measurement capabilities could be leveraged to provide benefits in the targeted proteomics domain as well. Here, we propose a new targeted proteomics paradigm centered on the use of next generation, quadrupole-equipped high resolution and accurate mass instruments: parallel reaction monitoring (PRM). In PRM, the third quadrupole of a triple quadrupole is substituted with a high resolution and accurate mass mass analyzer to permit the parallel detection of all target product ions in one, concerted high resolution mass analysis. We detail the analytical performance of the PRM method, using a quadrupole-equipped bench-top Orbitrap MS, and draw a performance comparison to selected reaction monitoring in terms of run-to-run reproducibility, dynamic range, and measurement accuracy. In addition to requiring minimal upfront method development and facilitating automated data analysis, PRM yielded quantitative data over a wider dynamic range than selected reaction monitoring in the presence of a yeast background matrix because of PRM's high selectivity in the mass-to-charge domain. With achievable linearity over the quantifiable dynamic range found to be statistically equal between the two methods, our investigation suggests that PRM will be a promising new addition to the quantitative proteomics toolbox.
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                Author and article information

                Journal
                24138574
                3833812
                10.1586/14737159.2013.845089
                Unknown

                biomarker discovery, diagnostics, digital maps, mass spectrometry,proteomics, reference maps, selected reaction monitoring,swath ms

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