PNNL

Abstract

Protein analysis of small numbers of human cells and single cells is primarily achieved by targeted proteomics with antibody-based immunoassays, whereas they have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative in terms of being antibody-free, high multiplex, high specificity, and quantitation accuracy. Recent advances in MS instrumentation makes MS-based targeted single-cell proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical issue for effective processing of single cells with minimal sample loss for MS analysis. To address this issue we have recently developed a simple protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) – selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells including single cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses and high-specificity LC-SRM to effectively address the increased dynamic concentration range resulted from the addition of exogeneous carrier protein. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. cLC-SRM was demonstrated to enable multiplexed accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. Most importantly, this method can be easily implemented in any MS and proteomics laboratories at no additional cost for instrument or reagents. Further improvements in detection sensitivity and sample throughput are needed towards multiplexed targeted single-cell proteomics analysis. We anticipate that eventually it can be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.