Single Cell Proteomics

Single-cell proteomics helped discover the earliest symmetry breaking during mammalian development.

https://www.cell.com/cell/fulltext/S0092-8674(25)01255-3

Multiplexed single-cell proteomics is getting a big boost 🚀

It's not just speed & scale ...
It's also data quality, depth, rich spectra improving de novo sequencing & accurate quantification.

Mass spectrometry-based proteomics enables comprehensive characterization of protein abundance, function, and interactions. Label-free approaches are simple to implement but challenging to scale to thousands of samples per day. Multiplexed techniques, such as plexDIA, can address these limitations but remain restricted by the lack of mass tags optimized for data-independent acquisition (DIA) workflows. Here, we present a systematic approach screening a library of 576 compounds that identifies several small molecules that, when conjugated to peptides, improve their detection and sequence identification by mass spectrometry. The lead molecule, PSMtag, substantially increases the detection of fragment b-ions, which increases the confidence of sequence identification and enhances de novo sequencing. PSMtags allow 9-plexDIA, using only stable isotopes of carbon, oxygen and nitrogen. As a result, it allows simultaneously increasing proteome coverage and sample throughput for plexDIA workflows without compromising quantitative accuracy. We demonstrate 240 samples-per-day with 9-plexDIA, while acquiring 28,359 protein data points in the same time label-free methods acquire 4,340. Our approach constitutes an expandable framework for designing mass tags to overcome existing limitations in multiplexed proteomics and provides plexDIA reagents capable of analyzing over 1,000 samples per day when using 10 minute runs. By facilitating higher throughput and improved identification, this innovation holds significant potential for accelerating proteomic studies across diverse biological and clinical applications.

PSMtags improve peptide sequencing and throughput in sensitive proteomics Mass spectrometry-based proteomics enables comprehensive characterization of protein abundance, function, and interactions. Label-free approaches are simple to implement but challenging to scale to thousands of samples per day. Multiplexed techniques, such as plexDIA, can address these limitations b...

One more week to submit abstracts !
https://single-cell.net/proteomics/scp2025 -and-submit-abstracts

Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP. Nat Protoc 19, 3750–3776 (2024).
https://www.nature.com/articles/s41596-024-01033-8.

Single-cell proteomics by mass spectrometry (MS) allows the quantification of proteins with high specificity and sensitivity. To increase its throughput, we developed nano-proteomic sample preparation (nPOP), a method for parallel preparation of thousands of single cells in nanoliter-volume droplets deposited on glass slides. Here, we describe its protocol with emphasis on its flexibility to prepare samples for different multiplexed MS methods. An implementation using the plexDIA MS multiplexing method, which uses non-isobaric mass tags to barcode peptides from different samples for data-independent acquisition, demonstrates accurate quantification of ~3,000–3,700 proteins per human cell. A separate implementation with isobaric mass tags and prioritized data acquisition demonstrates analysis of 1,827 single cells at a rate of >1,000 single cells per day at a depth of 800–1,200 proteins per human cell. The protocol is implemented by using a cell-dispensing and liquid-handling robot—the CellenONE instrument—and uses readily available consumables, which should facilitate broad adoption. nPOP can be applied to all samples that can be processed to a single-cell suspension. It takes 1 or 2 d to prepare >3,000 single cells. We provide metrics and software (the QuantQC R package) for quality control and data exploration. QuantQC supports the robust scaling of nPOP to higher plex reagents for achieving reliable and scalable single-cell proteomics.

https://doi.org/10.1038/s41596-024-01033-8

Join us to present, discuss & engage: single-cell.net

Submissions from early career researchers are strongly encouraged.

A discussion about proteomic technologies & the biology progress that they can enable.

They may unblock biology research.

Unveiling Proteins: The Future Of Single-Cell Proteomics With Professor Nikolai Slavov In this episode of FYI, Brett Winton, ARK's Chief Futurist, and Nemo Marjanovic, ARK's Multiomics Analyst, have an in-depth discussion with Professor Nikolai...

This week, we shared results from 2 projects. Each took over 5 years & they were published within an hour. This talk summarizes them.
1. Alternate RNA decoding results in stable and abundant proteins in mammals
2. Proteome asymmetry in mouse and human embryos before fate specification

Uncovering secrets of the proteome: Alternate RNA decoding & Protein asymmetry shaping cell fate This presentation "Uncovering the secrets of the human proteome" was given in May 2024 at the 50th anniversary celebration of the Barnett Institute by Prof. ...

As single-cell mass spec proteomics scales in throughput, depth & plex-ing, we need protocols to match. This Nature Protocol demonstrates ~ 4,000 single cell prepared / day and over 1,000 single-cell proteomes analzyed / day.

Single-cell proteomics by mass spectrometry (MS) allows the quantification of proteins with high specificity and sensitivity. To increase its throughput, we developed nano-proteomic sample preparation (nPOP), a method for parallel preparation of thousands of single cells in nanoliter-volume droplets deposited on glass slides. Here, we describe its protocol with emphasis on its flexibility to prepare samples for different multiplexed MS methods. An implementation using the plexDIA MS multiplexing method, which uses non-isobaric mass tags to barcode peptides from different samples for data-independent acquisition, demonstrates accurate quantification of ~3,000–3,700 proteins per human cell. A separate implementation with isobaric mass tags and prioritized data acquisition demonstrates analysis of 1,827 single cells at a rate of >1,000 single cells per day at a depth of 800–1,200 proteins per human cell. The protocol is implemented by using a cell-dispensing and liquid-handling robot—the CellenONE instrument—and uses readily available consumables, which should facilitate broad adoption. nPOP can be applied to all samples that can be processed to a single-cell suspension. It takes 1 or 2 d to prepare >3,000 single cells. We provide metrics and software (the QuantQC R package) for quality control and data exploration. QuantQC supports the robust scaling of nPOP to higher plex reagents for achieving reliable and scalable single-cell proteomics.

Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP - Nature Protocols nPOP is a method for parallel preparation of thousands of single cells in nanoliter-volume droplets deposited on glass slides by using the CellenONE instrument. This protocol describes the liquid handling for multiplexed mass spectrometry proteomics.