Single-cell proteomics capabilities have the potential to transform biomedical research and enable understanding of biological systems with a new level of granularity. Recent advances in sample ...processing, separations and MS instrumentation now make it possible to quantify >1000 proteins from individual mammalian cells.
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Highlights
•Label-free and isobaric labeling approaches for in-depth profiling of single cells.•Miniaturization and simplification of sample processing reduce surface losses.•Nanoflow separations enhance ionization efficiency and reduced chemical noise.•Ultrasensitive mass spectrometry and gas-phase separation add selectivity.
MS-based proteome profiling has become increasingly comprehensive and quantitative, yet a persistent shortcoming has been the relatively large samples required to achieve an in-depth measurement. Such bulk samples, typically comprising thousands of cells or more, provide a population average and obscure important cellular heterogeneity. Single-cell proteomics capabilities have the potential to transform biomedical research and enable understanding of biological systems with a new level of granularity. Recent advances in sample processing, separations and MS instrumentation now make it possible to quantify >1000 proteins from individual mammalian cells, a level of coverage that required an input of thousands of cells just a few years ago. This review discusses important factors and parameters that should be optimized across the workflow for single-cell and other low-input measurements. It also highlights recent developments that have advanced the field and opportunities for further development.
What’s new in single-cell proteomics Truong, Thy; Kelly, Ryan T.
Current opinion in biotechnology,
April 2024, 2024-04-00, 20240401, Letnik:
86
Journal Article
Recenzirano
In recent years, single-cell proteomics (SCP) has advanced significantly, enabling the analysis of thousands of proteins within single mammalian cells. This progress is driven by advances in ...experimental design, with maturing label-free and multiplexed methods, optimized sample preparation, and innovations in separation techniques, including ultra-low-flow nanoLC. These factors collectively contribute to improved sensitivity, throughput, and reproducibility. Cutting-edge mass spectrometry platforms and data acquisition approaches continue to play a critical role in enhancing data quality. Furthermore, the exploration of spatial proteomics with single-cell resolution offers significant promise for understanding cellular interactions, giving rise to various phenotypes. SCP has far-reaching applications in cancer research, biomarker discovery, and developmental biology. Here, we provide a critical review of recent advances in the field of SCP.
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•Recent advances in single-cell proteomics.•Improvements in sample preparation, separation techniques, mass spectrometry instrumentation, and data acquisition/analysis.•Prospects for further advances in sensitivity and throughput.
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.
Biological tissues exhibit complex spatial heterogeneity that directs the functions of multicellular organisms. Quantifying protein expression is essential for elucidating processes within complex ...biological assemblies. Imaging mass spectrometry (IMS) is a powerful emerging tool for mapping the spatial distribution of metabolites and lipids across tissue surfaces, but technical challenges have limited the application of IMS to the analysis of proteomes. Methods for probing the spatial distribution of the proteome have generally relied on the use of labels and/or antibodies, which limits multiplexing and requires a priori knowledge of protein targets. Past efforts to make spatially resolved proteome measurements across tissues have had limited spatial resolution and proteome coverage and have relied on manual workflows. Here, we demonstrate an automated approach to imaging that utilizes label-free nanoproteomics to analyze tissue voxels, generating quantitative cell-type-specific images for >2000 proteins with 100-µm spatial resolution across mouse uterine tissue sections preparing for blastocyst implantation.
Effective extension of mass spectrometry-based proteomics to single cells remains challenging. Herein we combined microfluidic nanodroplet technology with tandem mass tag (TMT) isobaric labeling to ...significantly improve analysis throughput and proteome coverage for single mammalian cells. Isobaric labeling facilitated multiplex analysis of single cell-sized protein quantities to a depth of ∼1 600 proteins with a median CV of 10.9% and correlation coefficient of 0.98. To demonstrate in-depth high throughput single cell analysis, the platform was applied to measure protein expression in 72 single cells from three murine cell populations (epithelial, immune, and endothelial cells) in <2 days instrument time with over 2 300 proteins identified. Principal component analysis grouped the single cells into three distinct populations based on protein expression with each population characterized by well-known cell-type specific markers. Our platform enables high throughput and unbiased characterization of single cell heterogeneity at the proteome level.
We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence‐activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing ...chip, after which samples were analyzed by ultrasensitive nanoLC‐MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single‐cell proteomics platform can be used to differentiate cell types from enzyme‐dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.
Single‐cell proteomics: A microfluidic platform coupled to nanoLC‐MS was developed to enable quantitative proteomic analysis of single mammalian cells containing only 0.1–0.2 ng of total protein. Label‐free cell differentiation was enabled by quantifying protein expression in individual cells.
Single-cell proteomics can provide unique insights into biological processes by resolving heterogeneity that is obscured by bulk measurements. Gains in the overall sensitivity and proteome coverage ...through improvements in sample processing and analysis increase the information content obtained from each cell, particularly for less abundant proteins. Here we report on improved single-cell proteome coverage through the combination of the previously developed nanoPOTS platform with further miniaturization of liquid chromatography (LC) separations and implementation of an ultrasensitive latest generation mass spectrometer. Following nanoPOTS sample preparation, protein digests from single cells were separated using a 20 μm i.d. in-house-packed nanoLC column. Separated peptides were ionized using an etched fused-silica emitter capable of stable operation at the ∼20 nL/min flow rate provided by the LC separation. Ultrasensitive LC–MS analysis was achieved using the Orbitrap Eclipse Tribrid mass spectrometer. An average of 362 protein groups were identified by tandem mass spectrometry (MS/MS) from single HeLa cells, and 874 protein groups were identified using the Match Between Runs feature of MaxQuant. This represents an >70% increase in label-free proteome coverage for single cells relative to previous efforts using larger bore (30 μm i.d.) LC columns coupled to a previous-generation Orbitrap Fusion Lumos mass spectrometer.
Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several techniques to prepare and analyze ...low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While being effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited implementation in most proteomic laboratories. Here, we report a fully automated platform termed autoPOTS (automated preparation in one pot for trace samples) that uses only commercially available instrumentation for sample processing and analysis. An unmodified, low-cost commercial robotic pipetting platform was utilized for one-pot sample preparation. We used low-volume 384-well plates and periodically added water or buffer to the microwells to compensate for limited evaporation during sample incubation. Prepared samples were analyzed directly from the well plate with a commercial autosampler that was modified with a 10-port valve for compatibility with 30 μm i.d. nanoLC columns. We used autoPOTS to analyze 1–500 HeLa cells and observed only a moderate reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. To evaluate clinical feasibility, we identified an average of 1095 protein groups from ∼130 sorted B or T lymphocytes. We anticipate that the straightforward implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.
We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass ...spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS analysis of multiply charged peptides, resulting in an average of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential analysis of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification.
The combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS) and latest-generation mass spectrometry instrumentation provides dramatically improved single-cell proteome profiling.
Mass spectrometry (MS)-based proteomics is emerging as a broadly effective means for identification, characterization, and quantification of proteins that are integral components of the processes ...essential for life. Characterization of proteins at the proteome and sub-proteome (e.g., the phosphoproteome, proteoglycome, or degradome/peptidome) levels provides a foundation for understanding fundamental aspects of biology. Emerging technologies such as ion mobility separations coupled with MS and microchip-based-proteome measurements combined with MS instrumentation and chromatographic separation techniques, such as nanoscale reversed phase liquid chromatography and capillary electrophoresis, show great promise for both broad undirected and targeted highly sensitive measurements. MS-based proteomics increasingly contribute to our understanding of the dynamics, interactions, and roles that proteins and peptides play, advancing our understanding of biology on a systems wide level for a wide range of applications including investigations of microbial communities, bioremediation, and human health.
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