U.S. Department of Energy

Pacific Northwest National Laboratory

A carrier-assisted targeted mass spectrometry approach for proteomics analysis of single cells

Introduction 

Antibody-based flow cytometry and mass cytometry are predominant technologies for targeted proteomics analysis of single cells. However, they share common shortcomings with other antibody-based methods (e.g., low-multiplex, the need of high-quality antibodies, and unavailability of antibodies for new proteins). Furthermore, they lack quantitation accuracy to provide accurate protein concentrations. Mass spectrometry (MS)-based targeted proteomics has emerged as a promising alternative for antibody-free precise high-multiplex quantification of target proteins. However, it does not meet the merit for analysis of single cells due to insufficient sensitivity. To tackle this issue, we recently developed a new targeted MS approach that couples carrier-assisted sample preparation to a highly sensitive targeted MS platform for enabling proteomics analysis of single cells.

Methods 

In complex biological samples low abundant proteins can be reliably detected without loss because highly abundant proteins severe as an effective carrier to prevent their loss. With this observation, we recently developed a simple preparation method with using exogenous BSA protein as a carrier for lossless processing of single cells. A highly sensitive targeted MS platform PRISM-SRM/PRM (high-pressure, high-resolution separations with intelligent selection and multiplexing coupled to selected/parallel reaction monitoring) was then used for absolute quantification of key EGFR pathway proteins in 1-100 HMEC cells. The Waters nanoACQUITY UPLC system and the Thermo Scientific TSQ Vantage or Q Exactive MS instrument were used for LC separation and targeted quantification, respectively. The Skyline software was employed for analysis of SRM/PRM data. 

Preliminary data 

Recently PRISM-SRM was demonstrated for enabling highly sensitive quantification of target proteins at ≤100 copies per human cell when starting with only ~25 µg of cell lysate digests, which is equal to ~250,000 human cells. However, in the real clinical applications the available sample amount could be much smaller (e.g., 1-10 circulating tumor cells) and sample loss is almost inevitable for current targeted MS analysis. With the introduction of exogenous proteins as a carrier to prevent sample loss we refined our PRISM termed carrier-assisted PRISM (i.e., cPRISM) for enabling targeted quantification of proteins in single cells by coupling with SRM/PRM. In our proof-of-concept experiment BSA protein was selected as a carrier for assisting processing of a small number of cells. 1-100 isolated HMEC cells were collected into 50 µg of BSA carrier-containing tubes to prevent cell adhesion to the tube wall. When mixed with BSA carrier, the small number of cells were processed as easily as bulk cells with minimal sample loss. The constant ‘unbiased’ peptide recovery was observed across all the samples with confident detection of multiple endogenous peptides with different hydrophobicity at 1-5 HMEC cells as well as the linear response curves of target proteins from 1 to 100 cells. This result has shown the carrier-assisted method can be used to effectively process single cells for highly sensitive PRISM-SRM quantification. Furthermore, detection of SFADINLYR derived from NRAS in small numbers of HMEC cells (~80,000 NRAS molecules per HMEC cell from bulk cells) suggested that our current targeted MS platforms can provided ~100 zmol level of sensitivity. Currently we are working on further improving cPRISM-SRM performance in sensitivity, throughput and robustness. We envision that this new targeted MS approach will have broad utilities in biomedical research (e.g., single cells and clinical applications with extremely small sample amounts).  

Novel aspect 

cPRISM-SRM/PRM is a simple, convenient targeted MS approach for highly sensitive quantification of proteins in single cells.

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