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Overview
Mass spectrometry (MS) offers the most unbiased approach of all the methods used for single-cell proteomics. MS enables the identification and quantitation of thousands of proteins, proteoforms, and post translational modifications (PTMs) in single cells.
The pillars of successful single-cell proteomics workflow require throughput, quantitation, sensitivity and coverage. By collaborating with the scientific community, we at Thermo Fisher Scientific have developed innovations that enable you to perform single-cell proteomics analysis to reveal the full complexity of cellular diversity. Our latest innovation in the Thermo Scientific Orbitrap family of mass spectrometers — the Thermo Scientific Orbitrap Astral Mass Spectrometer — provides the sensitivity and throughput needed to deliver more proteins per cell, >100s of cells per day from cell populations, cell types and states, on a cell-to-cell basis, generating maximum insights that assess cell heterogeneity and rare cell types.
Thermo Scientific Orbitrap mass spectrometers offer leading mass spectrometry technology for accurately identifying and quantifying 1000s of proteins with more than five orders of linear dynamic range and tight coefficients of variation right down to single cell level. Orbitrap mass spectrometers are highly sensitive to accurately record differences in proteins across individual cells and provide a meaningful view of their biological states.
Learn how the Orbitrap Astral Mass Spectrometer opens a new horizon for single-cell proteomics by empowering higher throughput and deeper coverage without sacrificing quantitative accuracy and precision.
Deeper proteome coverage and faster throughput for single-cell samples
The Orbitrap Astral Mass Spectrometer enables robust, reproducible, rapid, and sensitive deep proteome coverage from low sample amounts.
Thermo Scientific Single-Cell Proteomics Platform
Thermo Scientific Single-Cell Proteomics Platform meets the pillars of successful single-cell proteomics technology.
What do you get from Thermo Scientific Single-Cell Proteomics Platform.
Accurate and precise quantitation
- Determination of the correct protein abundance in a cell
- Confidant and accurate quantitation of low-abundance proteins
- Reduced number of measurements needed to draw conclusions
High confidence with deep proteome coverage and data completeness
- 100s-1000s of proteins from a single cell
- Wide dynamic range to discover differentially expressed low-abundance proteins of biological significance
- Orbitrap based DIA single-cell proteomics workflow provides >99% data completeness
- TMT single-cell proteomics workflow is ideal for mapping small protein changes from low-level signals
High throughput for large number of cells to observe biological insight
- Provides statistical power to infer biological patterns
Robust platform and method
- Easy day-to-day platform operation for analyses of thousands of cells
- Automated sample prep for DIA and TMT single-cell proteomics workflows
Workflows
High throughput DIA single-cell proteomics workflow for maximum proteome coverage
Thermo Scientific Orbitrap based DIA single-cell proteomics workflows provide sensitivity, quantitative accuracy and precision, in combination with high proteome coverage and data completeness, for high-confidence, high-throughput characterization of individual cells. The workflow requires minimum sample preparation and provides an unbiased approach for identifying diverse proteins from phenotypically distinct cells extracted from complex samples. Also, this workflow is better suited for characterizing proteomes from rare cells.
Deep coverage for limited sample and single cells at faster throughput
Hela Dilution experiments. 50pg to 5 ng injected on column and analyzed by Orbitrap Astral MS by either 72 SPD or 50 SPD methods.
Methods for throughput
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Methods for maximizing identification
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Single HeLa cells analyzed by Orbitrap Astral MS 72 SPD method
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"With the Orbitrap Astral mass spectrometer,we were able to double the throughput for single cells, while also increasing the number of proteins per cell by 50%. This means we start picking up key cellular decision making proteins like transcription factors, epigenetic regulators and kinases to better understand the biological heterogeneity of single cells."
— Erwin Schoof, Associate Professor, Department of Biotechnology and Biomedicine, Section for Protein Science and Biotherapeutics, Cell Diversity Lab, Technical University of Denmark
"With the Orbitrap Astral mass spectrometer we're pushing boundaries with a nearly lossless label free method. Our workflow reveals >5000 proteins and ~40,000 peptides per HeLa cell, setting new standards in SCP! The great depth enabled direct detection of PTMs in single cells, eliminating the need for prior enrichment. We've uncovered extensive phosphorylation and glycosylation in single-cell samples."
— Jesper Olsen, Deputy Center Director, Professor, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen
Higher throughput TMT multiplexing single-cell proteomics workflow for high quantitative precision and accuracy
Thermo Scientific TMT multiplexing provides the highest throughput of any single-cell proteomics workflow along with high quantitative precision and accuracy. Additionally, the use of TMT multiplexing enables the use of any Orbitrap based mass spectrometer for single-cell proteomics by boosting the signal of the peptides for detection and sequencing by mass spectrometry.
Single cell throughput using TMT multiplexing
"Thanks to Orbitrap MS and TMTpro technology, we can in principle multiplex 18 single cells at the same time. With advanced chromatography, this enables the possibility of running multiple 10,000-cell projects per instrument, per year, leading to real impactful biological insights across life science research."
— Erwin Schoof, Associate Professor, Department of Biotechnology and Biomedicine, Section for Protein Science and Biotherapeutics, Cell Diversity Lab, Technical University of Denmark
Products
Sample preparation optimized for single-cell proteomics
Manual sample preparation is not feasible for single-cell proteomics due to limited starting material and the shear number of cells to be analyzed. Automation addresses many of the short comings of single cell sample preparation. From cell sorting and isolation to preparation of proteins for mass spectrometry.
For single cell sorting, isolation, nanoliter dispensing and sample preparation for MS
cellenONE® platform combines high accuracy single cell isolation and precision nanoliter reagent dispensing, allowing for miniaturization of single cell proteomics protocols and enabling automated sample preparation for label-free and TMT multiplexing experiments.
- Single-cell sorting and isolation
- Any cell type
- Protein digestion for DIA based single-cell proteomics
- Protein digestion and labeling for TMT based single-cell proteomics
Mass Spectrometers for single-cell proteomics
With the Orbitrap mass spectrometer at the heart of the single-cell proteomics workflow, we can obtain a wealth of information inside every cell.
For experiments that require thousands of cells to gain real biological insights
With an unprecedentedly low detection level, the Thermo Scientific Orbitrap Astral Mass Spectrometer accesses more proteins from a single cell than ever before. And with the ability to multiplex single cells at a higher throughput, hundreds of single cells can be quantitatively analyzed in a single day, allowing researchers to efficiently and reliably determine the heterogeneity of a cell’s biology, unlocking never-before-seen insights that can revolutionize research.
Research level single-cell proteomics system for accurate and precise quantitation
TMT multiplexing workflow provides the highest throughput for single-cell proteomics. The SPS MS3 method on the Thermo Scientific Orbitrap Ascend MultiOmics Tribrid Mass Spectrometer provides the highest accuracy and precision for TMT-based quantitation throughout wide concentration range. The real-time search acquisition strategy on this platform improves the ratio accuracy for TMT reporter ions, boosts sensitivity and increases total quantified peptides. The Orbitrap Ascend Tribrid MS is also excellent for single-cell proteomics DIA workflow for those researchers want that capability as well.
Entry level single-cell proteomics system
The single-cell proteomics DIA workflow on the Thermo Scientific Orbitrap Exploris 480 Mass Spectrometer offers research laboratories accurate and precise quantitation, with deep proteome coverage and data completeness. For laboratories wanting to perform TMT based single-cell proteomics workflow, the Orbitrap Exploris 480 MS is versatile enough to handle that as well. The TurboTMT intelligent acquisition available on the mass spectrometer speeds up spectral acquisition for TMT 18plex experiments, thereby providing the same throughput for single-cell proteomics experiments that Orbitrap Tribrid mass spectrometers provide.
 Thermo Scientific Orbitrap Astral Mass Spectrometer |  Thermo Scientific Orbitrap Ascend MultiOmics Tribrid Mass Spectrometer |  Thermo Scientific Orbitrap Exploris 480 Mass Spectrometer | |
|---|---|---|---|
| Ideal Workflows | DIA Single-cell proteomics TMT Single-cell proteomics | DIA Single-cell proteomics TMT Single-cell proteomics | DIA Single-cell proteomics |
| Ideal Study | >1000 cells study with TMT <1000 cells study with DIA | >1000 cells study with TMT <500 cells study with DIA | <500 cells study with DIA |
| DIA Single-cell proteomics Throughput | 100 cells/day | 100 cells/day | 100 cells/day |
| TMT Single-cell proteomics Throughput | 500 cells/day | 200 cells/day | 200 cells/day |
| DIA Single-cell proteomics Quantitative Accuracy |  |  | |
| TMT Single-cell proteomics Quantitative Accuracy |  | | |
| DIA Single-cell proteomics Quantitative Precision | | | |
| TMT Single-cell proteomics Quantitative Precision | | | |
| DIA Single-cell proteomics Proteome Coverage | | | |
| TMT Single-cell proteomics Proteome Coverage | | | |
| Single-Cell sensitivity | | | |
| = Good | = Better | = Best |
Separations optimized for single-cell proteomics
Every cell counts and separations of peptides are crucial in single-cell proteomics. We’ve taken steps to optimize HPLC, column chemistries and differential ion mobility devices to enable studies that require in depth analysis to short high throughout applications.
Vanquish Neo UHPLC System
The Thermo Scientific Vanquish Neo UHPLC System is designed for high-sensitivity LC-MS applications such as single-cell proteomics. It flawlessly integrates with industry-leading Thermo Scientific mass spectrometry portfolio and ESI sources.
50 cm µPAC Neo HPLC Columns
The 50 cm µPAC Neo HPLC Columns are designed to get maximum proteome coverage from single-cell to sub nanogram protein digest samples.
FAIMS Pro Duo Interface
The FAIMS Pro Duo Interface provides enhanced selectivity and sensitivity down to at a low nanogram to single-cell level allowing for deeper proteome coverage.
Reagents for single-cell proteomics
The Thermo Scientific Tandem Mass Tag (TMT) reagents are designed to increase the level of cell multiplexing. Currently, the TMT enabled workflow provides the highest throughput for single-cell proteomics experiments.
TMTpro 18-plex Label Reagents
TMTpro 18-plex Label Reagents are designed for the next level of sample multiplexing. TMTpro 18-plex enables multiplexing up to 18 cells, enabling greater throughput for protein identification and quantitative analysis by mass spectrometry.
Data analysis for single-cell proteomics
Optimized for data generated from single-cell proteomics regardless of the workflow undertaken (TMT or DIA).
Proteome Discoverer Software
Thermo Scientific Proteome Discoverer Software offers predefined workflows to support both MS2 and MS3-based TMT quantification. The CHIMERYS™ intelligent search algorithm by MSAID available within Proteome Discoverer software enables processing of DIA data.
Webinars
Listen to some of the leaders in the field of integrative structural biology discuss how integrative structural biology is making a difference in their work.
Innovations in single-cell and deep visual proteomics
Making single-cell proteomics biologically relevant
Sophia Steigerwald, Max-Planck Institute
Manuel Matzinger, Proteomics Technology Hub, IMP, Vienna, Austria
Karl Mechtler, Proteomics Technology Hub, IMP, Vienna, Austria
The webinar will focus specifically on single-cell proteomics and deep visual proteomics and how latest innovations in mass spectrometry, namely the Thermo Scientific Orbitrap Astral MS, has helped advance the fields.
Single-cell proteomics for the exploration of cell heterogeneity
Erwin Schoof, DTU Proteomics
Learn more about recent innovations in single-cell proteomics. Advances in instrumentation, including the novel Orbitrap Astral MS, and how to use both TMTPro-labeled and label-free approaches.
Best Practices for Single-Cell Proteomics Using Mass Spectrometry
Fernando Salvato, Thermo Fisher Scientific
Runsheng Zheng, Thermo Fisher Scientific
Hear best practices for performing DIA-based single-cell proteomics using Orbitrap mass spectrometers.
Antigenic T-cell Response Profiling in COVID-19 Using Single-Cell Proteomics and Transcriptomics
Benedikt Kessler, University of Oxford
View the Antigenic T-cell Response Profiling in COVID-19, employing both single-cell proteomics and transcriptomics in NP-specific T-cells from acute SARS-CoV-2 patients.
Single-cell multi-omics: Next steps
Bogdan Budnik, Wyss Institute at Harvard University
Dr. Budnik will present an LC-MS workflow that allows researchers to perform proteomic and metabolomic analysis on the same type of chromatography simultaneously in parallel. Additionally, transcriptomic and genomic analysis can be added as extra options to get a “multi-omic analysis of the single cell” that captures the full biological state of the cell based on all critical components of cellular expression.
One Cell at a Time: Single Cell Proteomics Becomes a Reality
Ryan Kelly, Brigham Young University
This webinar will describe: nanoPOTS, a new sample processing platform to maximize sample recovery; miniaturization of LC separations; and latest-generation of MS instrumentation to provide the required sensitivity and throughput for effective analysis of trace samples.
Multiplexing Single Cell Proteomics: A Marriage of Sensitivity and Throughput
Erwin Schoof, DTU Proteomics
See how a 384-well workflow, using standard off-the-shelf components, allows characterization of single cells to a protein depth of well over 1,000 proteins per cell.
Using Single-Cell Proteomics and Transcriptomics to Understand Gene Regulation
Nikolai Slavov, Northeastern University
Learn about a new method for single-cell proteomics for increased accuracy and throughput while lowering cost time for the analysis of cellular heterogeneity in homogeneous monocytes.
Pushing the limits of multiplexed single-cell proteomics
David Perlman, Biopharma Consultant
This webcast will cover a roadmap for single-cell proteomics parameter optimization for human immune cell profiling using the tandem mass tag (TMT) multiplexed SCoPE-MS methodology.
New development in mass spectrometry-based single-cell proteomics
Karl Mechtler, IMP, Vienna BioCenter
This webinar will present the use of a single-cell proteomics sample preparation device, coupled to mass spectrometer to increase sensitivity and throughput.
Enhancing limited sample- and single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography
Pedro Aragón Fernández, Technical University of Denmark
This webinar will highlight limited sample- and single-cell proteomics through tailored DIA and micropillar array-based chromatography.
Resources
Go beyond—Limited samples to single-cell proteomics
Leading the way in single-cell proteomics means collaborating with experts in the scientific community. By joining forces with single-cell proteomics leaders from around the world, we have taken a holistic approach to advancing this field. We’ve developed innovations in single-cell proteomics that allow you to move away from bulk sample to rare cells, single cells and spatial proteomics that reveal the full complexity of cellular diversity.
Challenges and emerging directions in single-cell proteomics
The evolution of modern single-cell proteomics
Optimized one-pot single-cell proteomics workflow
Single-Cell Proteomics Publications
Single-cell sensitivity
Deeper proteome coverage and faster throughput
Single-Cell Proteomics on Orbitrap Astral MS
Using the proteoCHIP 12*16 autosampler holder for the Vanquish Neo UHPLC system
Style Sheet for Global Design System
Style Sheet for Komodo Tabs
CMD Wide-format style fixes