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. Author manuscript; available in PMC: 2021 Aug 7.
Published in final edited form as: J Vis Exp. 2020 Nov 6;(165):10.3791/61797. doi: 10.3791/61797

Figure 1: Overview of all the steps in cLC-SRM (carried-assisted one-pot sample preparation coupled with liquid chromatography – selected reaction monitoring).

Figure 1:

Nonhuman cell lysate digests (e.g., S. oneidensis) is used to pretreat PCR tubes for coating tube surface. Small numbers of human cells or single cells sorted by FACS are collected into pretreated PCR tubes. BSA protein (or nonhuman cell lysate proteome) carrier, heavy internal standards, and TFE (or DDM) are added into sample tubes sequentially for facilitating cell lysis and reducing surface adsorption losses. Conceptually the combined DDM and nonhuman cell lysate proteome carrier will work well for cLC-SRM. Cell lysis is conducted by sonication and protein denaturation is achieved by heating at high temperature. DDT and IAA reagents are used for reduction and alkylation, respectively (the step is optional). Trypsin is added for digestion with much higher ratios of trypsin: protein than that for standard trypsin digestion. The cap of sample tube is removed and then PCR tube is inserted into LC vial for direct LC-SRM analysis. Collected SRM data are analysis using publicly available Skyline software.