Robust, sensitive, and quantitative single-cell proteomics based on ion mobility filtering

Abstract

Unbiased single-cell proteomics (scProteomics) promises to advance our understanding of the cellular composition of complex biological systems. However, a major challenge for current methods is their ability to identify and provide accurate quantitative information for low abundance proteins. Herein, we describe an ion mobility-enhanced mass spectrometry acquisition method, TIFF (Transferring Identification based on FAIMS Filtering), designed to improve the sensitivity and accuracy of label-free scProteomics. The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells with >1,100 proteins consistently quantified, a significant improvement in overall performance. We applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during a lipopolysaccharide stimulation experiment and uncovered unanticipated temporal response trajectories. Further, we demonstrated, to our knowledge, the first application of scProteomics to classify cell populations of a human organ (the lung) without prior antibody labeling.

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

“…Most reported mass-spectrometry-based scProteomics technologies can be classified based upon whether they make use of isotopic labeling; i.e., they are either label-free or use isobaric labeling. In the label-free methods 5 – 10 , single cells are individually processed and analyzed with liquid chromatography-mass spectrometry (LC-MS). Ion current measurements (i.e., MS1 ion currents) are used to quantify protein abundance.…”

To the proteome and beyond: advances in single-cell omics profiling for plant systems

“…On the MS side, gas-phase fractionation using ion mobility spectrometry (IMS) devices coupled to traditional LC–MS setups has proven to substantially increase ion selectivity and sensitivity by filtering out nonpeptide ions prior to MS analysis ( Cong et al, 2020 ; Greguš et al, 2020 ; Brunner et al, 2021 ; Stejskal et al, 2021 ; Woo et al, 2021a ). Using Nanodroplet Processing in One pot for Trace Sample (nanoPOTS) (see “Automated processing for small-volume samples”) sample preparation and optimized settings on a high field asymmetric IMS interface coupled to an Orbitrap Eclipse Tribrid MS identified over 1,000 protein groups from single HeLa and motor neuron cells ( Cong et al, 2020 ).…”

Proteomic/transcriptomic analysis of erythropoiesis

“…Notably, the Slavov laboratory recently demonstrated that a handful of TFs could by identified and quantified by sc-MS in macrophages [58 ▪▪ ]. Finally, temporal trajectories were recently established from sc-MS data of murine macrophages during lipopolysaccharide stimulation [60]. Based on these new exciting results, it is entirely conceivable that, with further advances in sample processing, experimental design, and computational approaches, MS-based proteomics will soon permit large-scale proteome measurements in single cells, opening new doors to understanding transcriptional regulation of erythropoiesis and other dynamic cellular processes.…”