Proteomic Sample Preparation through Extraction by Unspecific Adsorption on Silica Beads for ArgC-like Digestion
journal contributionposted on 30.01.2019, 00:00 by Yannik Lewin, Moritz Neupärtl, Vahid Golghalyani, Michael Karas
Sample preparation for mass-spectrometry-based proteomic analyses usually requires intricate, multistep workflows that are often limited in capacity or suffer from sample loss. Here, we introduce a lean adsorption-based protocol (ABP) for the extraction of proteins from fresh cell lysates that enables us to modify and tag protein samples under harsh conditions, such as organic solvents, high salt concentrations, or low pH values. This offers high versatility while also reducing the required steps in the preparation process significantly. Protein identifications are slightly increased compared to traditional acetone precipitation followed by an in-solution digestion (AP/IS) or filter aided sample preparation (FASP) and proved complementary to both methods regarding proteome coverage. When combined with ArgC-like digestion, this approach delivered 5386 uniquely identified proteins, a substantial increase of 18.27% over tryptic digestion (4554), while decreasing spectra complexity due to a lower number of peptide to spectra matches per protein and the number of missed cleaved peptides. In addition, an increased number of identified membrane proteins and histones as well as improved fragmentation and intensity coverage were observed through comprehensive data analysis.
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ArgC-like digestionpreparation processmultistep workflowsspectra complexitypeptidein-solution digestionintensity coveragetag protein samplesUnspecific Adsorptionmembrane proteinscell lysatesdata analysismass-spectrometry-based proteomic analysesproteome coveragesample lossFASPSilica BeadsArgC-like Digestion Sample preparationProtein identificationsProteomic Sample PreparationpH valuesadsorption-based protocolsalt concentrationsABPAPsample preparationacetone precipitation