Facile carrier-assisted targeted mass spectrometric approach for proteomic analysis of low numbers of mammalian cells

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.

Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt

“…Pairing the fluorescence-activated cell sorting (FACS) protocol with new methods for global proteome analysis of small numbers of cells (<200 cells) enabled us to compare the transcriptome and proteome for all six cell populations 27,28 (Supplementary Data #2). Despite the use of small cell numbers, particularly, for the rare EEC and tuft cell populations, we were able to quantify the expression of over 2,800 proteins and investigate RNA:protein correlation patterns ( Supplementary Figs.…”

Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells

“…We have recently demonstrated that the addition of exogenous carrier proteins allows small numbers of mammalian cells to be effectively processed using conventional sample preparation protocols with minimal sample loss. 41 However, our previous approach required high-resolution PRISM to reduce the increased sample dynamic concentration range resulting from the addition of large amounts of carrier protein, thus generating many fraction samples that significantly lowered sample throughput. To alleviate this problem, we have developed a carrier-assisted LC–SRM approach for precise multiplexed analysis of small numbers of mammalian cells.…”

“…For example, we have recently 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 low numbers of cells. 41 For cPRISM-SRM, we used bovine serum albumin (BSA) (~50 μ g) as a carrier to minimize losses in conjunction with highly sensitive PRISM-SRM and found that we could detect low-abundance proteins in as few as 100 mammalian cells as well as high- to moderate-abundance proteins in single mammalian cell equivalents. This is ~3–4 orders of magnitude lower than the cell number required for current targeted MS methods (typically ~10 5 –10 6 cells).…”

Clinical potential of mass spectrometry-based proteogenomics

“…The many benefits of targeted MS-based assays described above (precision, harmonization, multiplexing, specificity) need to be weighed against the limitations of this platform. For example, while coupling immuno-affinity enrichment of peptides to targeted MS provides enhanced sensitivity for detection of proteins in clinical samples, the sensitivity of immuno-MS assays is currently not sufficient to support single cell analyses, although progress is being made towards this goal 125,126 . Unlike single cell techniques (e.g., immunohistochemistry, flow cytometry), MS-based assays quantify the average amount of peptides and proteins across thousands of cells, similar to other extraction-based techniques such as ELISA and Western blotting.…”