Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

Abstract

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.

Nanowell-mediated two-dimensional liquid chromatography enables deep proteome profiling of <1000 mammalian cells

“…The preparation of the cellular material to produce ready-to-analyze peptides utilized our recently developed nanoPOTS method (Fig. 1a) on a microfluidic glass chip (chip 1) 24. While not the focus of this study, this nanoPOTS platform provides efficient processing of small biological samples into ready-to-analyze peptides.…”

“…The reconstituted volume was aspirated into the capillary and washed twice with 200 nL of 0.1% formic acid. The rinse solutions were also aspirated into the capillary as described previously 24. The capillary sections with collected fractions were sealed with Parafilm on both ends and stored at –20 °C for the second dimension low-pH nanoLC separation.…”

Transformative Opportunities for Single-Cell Proteomics

“…25,26 Even with mammalian cells having diameter ∼15 μ m, ESI−MS has made progress in quantifying increasing number of proteins in relatively small number of cells, reaching thousands of proteins in cell lysates corresponding to hundreds of cells 27 or even fewer cells. 28 Recently, we reported a method that allows quantifying over a thousand proteins across many single mouse stem cells. 20 We believe that the field is ready to take off from this launching point and increase the number of accurately quantified proteins and the number of single cells assayed by orders of magnitude.…”

“…To mimic that approach for single-cell proteomics, given the ∼500 pg of protein in a typical mammalian cell, 29 the reaction volume should be limited to nanoliters. Just recently, proteomics preparations have been scaled down to hundreds of nano-liters, 28 whereas single-cell transcriptomics often uses lysis volumes that are 100 times smaller, on the order of just a few nanoliters. 13 Protein losses can be further reduced by passivating the surfaces interfacing with the samples.…”

Single cell protein analysis for systems biology

“…Though current MS-based methods are limited in cell throughput to ~10 cells/h per instrument, incorporating existing automation technologies and increasing cellular multiplexing can increase cell throughput, a critical determinant of statistical power [22]. Reducing sample volumes from microliters to nanoliters can significantly alleviate protein adsorption, as demonstrated recently by lysing cells in hundreds of nanoliters using a custom chip dubbed ’nanodroplet processing in one pot for trace sample’ (nanoPOTS) [72]. Another avenue for improvement is peptide separation.…”