Description

Nanoliter One-Pot Sample Preparation Enables Deep, Global Proteome Profiling of Single Mammalian Cells

Ryan T. Kelly^1*, Ying Zhu¹, Paul D. Piehowski², Rui Zhao¹, Yufeng Shen², Ronald J. Moore², Wei-Jun Qian², Richard D. Smith²

¹Pacific Northwest National Laboratory (PNNL) Environmental Molecular Sciences Laboratory, ²PNNL Biological Sciences Division

Despite the power of mass spectrometry (MS)-based proteomics for understanding cellular function, the large amounts of material required for in-depth studies (typically thousands to millions of cells) hinder many applications. Analytical platforms based on ultrasensitive liquid chromatography coupled to MS are not the bottleneck, as single-cell sized samples have been accessible for over a decade. Rather, sample preparation procedures have been incapable of preparing and delivering such small samples to these analytical platforms. Conventional procedures based on pipetting and multiwell plates suffer from large sample losses due to adsorption to surfaces and poor reaction kinetics, such that orders of magnitude more starting material are required than should otherwise be necessary. We have developed NanoPOTS: Nanoliter Preparation in One pot for Trace Samples, in which a robotic platform dispenses cells and reagents into photolithographically patterned “nanopot” reaction vessels with subnanoliter precision. Sample preparation utilizes a novel workflow that eliminates the need for multiple reaction vessels and cleanup steps to process cellular tissue into purified tryptic peptides. Compared to the typical tens-of-microliter volumes for proteomic sample preparation, the ~200 nL nanowells minimize sample losses to surfaces and maintain elevated sample concentrations for efficient digestion. Samples comprising 10 to 140 HeLa cells were initially processed and analyzed using the NanoPOTS platform. More than 3100 proteins could be confidently and quantitatively identified for the smallest cell loading of ~10 cells, which is a degree of proteome coverage that has not been reported previously for fewer than 10,000 cells. Aliquots of the ~10-cell samples containing less protein than that of a single cell were also analyzed. >1,800 proteins were identified in these samples, indicating the immediate applicability of the NanoPOTS platform for deep, quantitative global single cell analysis.