American Chemical Society
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Two-Dimensional Peptide Separation Improving Sensitivity of Selected Reaction Monitoring-Based Quantitative Proteomics in Mouse Liver Tissue: Comparing Off-Gel Electrophoresis and Strong Cation Exchange Chromatography

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journal contribution
posted on 2016-02-20, 08:51 authored by Alexander Schäfer, Christine von Toerne, Silke Becker, Hakan Sarioglu, Susanne Neschen, Melanie Kahle, Stefanie M. Hauck, Marius Ueffing
Protein expression analysis is one of the most powerful tools to further the understanding of biological systems. Progress in the field of mass spectrometry has shifted focus from gel-based approaches to the upcoming LC-selected reaction monitoring (SRM) technique which combines high technical accuracy with absolute quantification of proteins and the capability for high-throughput analyses. Due to these properties, LC-SRM has the potential to become the foundation for biomarker analysis, targeted hypothesis driven proteomic studies and contribute to the field of systems biology. While the performance of LC-SRM applied to samples from various bodily fluids, particularly plasma, and microorganisms has been extensively investigated, there is only little experience with its application to animal tissue samples. Here, we show that a conventional one-dimensional LC-SRM workflow applied to mouse liver tissue suffers from a shortcoming in terms of sensitivity for lower abundance proteins. This problem could be solved through the extension of the standard workflow by an additional dimension of separation at the peptide level prior to online LC-SRM. For this purpose, we used off-gel electrophoresis (OGE) which is also shown to outperform strong cation exchange (SCX) in terms of resolution, gain of signal intensity, and predictability of separation. The extension of the SRM workflow by a high resolving peptide separation technique is an ideal combination as it allows the addition of stable isotope standards directly after trytic digestion and will increase the dynamic range of protein abundances amenable by SRM in animal tissue.