Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis

Abstract

Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic analysis in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (>99%), thus substantially increasing the analytical sensitivity for single cell samples. We applied the present system in the proteomic analysis of 100 ± 10, 50 ± 5, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, respectively. The OAD chip-based system was further applied in single mouse oocyte analysis, with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymatic digestion efficiency over the traditional in-tube system.

Microfluidic Single‐Cell Omics Analysis

“…C) A nanoliter‐scale oil‐air‐droplet (OAD) chip to conduct multistep complex sample pretreatment for LC‐MS/MS shotgun proteomic analysis. Reproduced with permission . Copyright 2018, American Society Chemistry.…”

“…Stationary nanoliter oil‐air‐droplet microreactors and manipulation techniques provided minimum sample loss and high‐efficient sample injection, thus increasing the analysis sensitivity substantially and enabling single‐cell analysis. By measuring single mouse oocyte proteome, OAD chip‐based system was verified to have advantages in sequence coverage, analyzing hydrophobic proteins and enzymatic digestion efficiency (Figure C) …”

Mass Spectrometry Advances and Perspectives for the Characterization of Emerging Adoptive Cell Therapies

“…Using this concept, Lombard and co-workers extended MS to probe the proteome of single cells, with subcellular resolution for embryonic cells from X. laevis and zebrafish embryos [111] and mouse neurons [112]. Using nanoliter-scale oil air droplet (OAD) chip, single HeLa cells and mouse oocytes were prepared for bottom-up analysis on an orbitrap instrument leading to the identification of~40 to~250 proteins on average, respectively [113]. Zhu et al further downscaled and partially automated the process for proteomic sample handling with the development of the nanodroplet processing in one-pot for trace proteomic samples (nanoPOTs) ( Figure 6) [88,114].…”

“…oocytes were prepared for bottom-up analysis on an orbitrap instrument leading to the identification of ~40 to ~250 proteins on average, respectively [113]. Zhu et al further downscaled and partially automated the process for proteomic sample handling with the development of the nanodroplet processing in one-pot for trace proteomic samples (nanoPOTs) ( Figure 6) [88,114].…”

Single-cell Analysis with Microfluidic Devices

“…We also used this strategy to identify functional proteins in the lysosome of tumors in another work. 47 More recently, Fang et al 48 described a nanoliter-scale oil-airdroplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single-cell proteomic analysis in the shotgun mode. As pioneers in the field of proteome analysis, Love et al [49][50][51] have reported a series of research work on the detection of cytokine secreting from single cells.…”