Abstract
There is an unmet technical challenge for mass spectrometry (MS)-based proteomic analysis of single mammalian cells. Quantitative proteomic analysis of single cells has been previously achieved by antibody-based immunoassays but is limited by the availability of high-quality antibodies. Herein we report a facile targeted MS-based proteomics method, termed cPRISM-SRM (carrier-assisted high-pressure, high-resolution separations with intelligent selection and multiplexing coupled to selected reaction monitoring), for reliable analysis of low numbers of mammalian cells. The method capitalizes on using “carrier protein” to assist processing of low numbers of cells with minimal loss, high-resolution PRISM separation for target peptide enrichment, and sensitive SRM for protein quantification. We have demonstrated that cPRISM-SRM has sufficient sensitivity to quantify proteins expressed at ≥200,000 copies per cell at the single-cell level and ≥3000 copies per cell in 100 mammalian cells. We envision that with further improvement cPRISM-SRM has the potential to move toward targeted MS-based single-cell proteomics.
Highlights
There is an unmet technical challenge for mass spectrometry (MS)-based proteomic analysis of single mammalian cells
When sample size becomes smaller, there is increasingly substantial and unavoidable loss through contact-surface adsorption regardless of current sample preparation methods[28,31]. To address this issue we developed a facile targeted mass spectrometric approach, termed cPRISM-SRM, for enabling proteomic analysis of very low numbers of mammalian cells. cPRISM-SRM capitalizes on the use of excessive exogenous protein as a carrier to minimize sample loss together with our recently developed high-resolution PRISM32 method to reduce the wide dynamic range of protein concentrations caused by the addition of protein carrier. cPRISM-SRM uses a sensitivetargeted MS platform (e.g., SRM)[10,33] for proteomic analysis of few cells
The development of cPRISM-SRM was inspired by our observation of reliable MS detection of extremely low-abundance proteins through extensive fractionation, apparently because highabundance proteins have served as an effective carrier to prevent their loss[37]
Summary
There is an unmet technical challenge for mass spectrometry (MS)-based proteomic analysis of single mammalian cells. Singlecell proteomics measurements exclusively rely on antibody-based immunoassays for targeted proteomic analysis of single cells[5,8] They have inherent limitations (e.g., low multiplex and enormous challenges of generating high-specificity antibodies, especially for protein mutations and posttranslational modifications). When sample size becomes smaller (close to single cells), there is increasingly substantial and unavoidable loss through contact-surface adsorption regardless of current sample preparation methods[28,31] To address this issue we developed a facile targeted mass spectrometric approach, termed cPRISM-SRM (carrier-assisted high-pressure, high-resolution separations with intelligent selection and multiplexing coupled to selected reaction monitoring), for enabling proteomic analysis of very low numbers of mammalian cells. We have shown that cPRISM-SRM enables detection of high- to moderate-abundance proteins in single HMEC cell equivalents and low-abundance proteins in ~100 HMEC cell equivalents, ~3–4 orders of magnitude lower than the cell number required for current targeted MS methods (typically ~105–106 cells[32,37])
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