The main goal of the new HUPO single-cell proteomics (SCP) initiative is to provide guidance and create a comparative overview of novel mass spectrometry (MS)-based low-input and single-cell technologies. After the first reports of SCP with large cells (i.e., oocytes) the field has quickly moved to ever more comprehensive analysis of single mammalian cells or even subcellular components.1–3 A variety of workflows have since demonstrated valuable insights into cellular identities and their function, driven entirely by the proteome and their post-translational modifications.4–6 However, deep proteome profiling of single mammalian cells or small subpopulations remains an analytical challenge for most non-specialized laboratories.
The growing interest in the SCP workflows and its application to biological questions has driven the initiation of this international SCP initiative. Our team is comprised of a diverse group of early, middle and advanced level scientists with growing expertise in diverse workflows and instrumentation. We aim to provide a comparative overview of current SCP approaches for the community to ease the implementation in more laboratories and integration to novel biological projects. Considering the ongoing trend away from bulk analysis towards resolving cellular heterogeneity, we believe that such unifying efforts will drive the understanding of complex tissue hierarchies and orchestrated spatiotemporal interactions.
To this end, our first goal is to perform a multi-laboratory study, processing the same batch of cells across different platforms and establish the first of such direct comparisons. We will leverage the knowledge of our SCP initiative members, who have pioneered various parts of the SCP workflows, including sample collection and preparation, chromatographic separation, acquisition strategies and analysis approaches.2,3,7–9 We consider utilization of one cell batch especially important for these comparisons, as cell handling and the cell cycle heavily influence cell size and proteome composition.5,10 Our portfolio of SCP workflows will include isobaric multiplexing (i.e. TMT) with one chemical label for multiple cells (i.e. carrier - SCoPE) to facilitate sufficient peptide signal even with less sensitive MS instrumentation.2 Following tremendous improvements in instrument sensitivity and implementation of ion-mobility separation also label-free SCP workflows have been successfully demonstrated.5,11 Those are typically paired with data independent acquisition (DIA), which massively reduces precursor stochasticity in comparison to data dependent acquisition.12 Most standard TMT-based workflows rely on small isolation windows to reduce precursor co-isolation to relatively quantify a single precursor through their reporter ions.13 Scanning speeds of current instrumentation therefore restrict isobaric labeling workflows to DDA, which suffer from detrimental amounts of missing data across large sample cohorts.12 The alternative label-free SCP approaches, however, are notoriously low in throughput as only once cell at a time is being analyzed in comparison to the isobaric labelling strategies (i.e. TMTpro = 18 samples or HyperSCP = 28).14,15 As most applications for SCP require the analysis of hundreds or even thousands of cells, most recently a combination of non-isobaric chemical labeling with DIA allowing for relative quantification between up to three samples at the MS1 level was introduced.16,17 This reduces missingness across a large number of samples while still increasing throughput through multiplexing.
As an alternative to chemical multiplexing, many groups aim to increase measurement throughput by optimizing chromatographic separation. Due to their low input, SCP samples do not require such extensive separation, allowing for the effective gradient length to be reduced in comparison to bulk.5,18–22 Short gradients and most efficient inject-to-inject times has been implemented with the disposable trap columns of the Evosep One. Those are regularly used for SCP workflows, due to the combination of in-line clean up the sample and simultaneous separation of peptides, while the previous sample is still acquired on the MS.5,23,24 SCP initiative team members also most recently demonstrated significantly reduced separation times employing non-commercial alternatives to increase chromatographic throughput.18,21,22 Additionally, to the various acquisition and peptide separation efforts, the SCP community has proposed smart strategy to streamline sample preparation for reduced peptide losses and improved reproducibility.2,7,8,11,20,25–27 Our comparative analysis will therefore include both plate-based sample preparation at higher volumes (i.e. 1-3uL) and dedicated chip or slide based approaches with low nanoliter volumes within dedicated instruments such as the cellenONE and Tecan Uno.3,7,20,27–29
The entire proteomics community, but especially researchers focusing on improvements in SCP, are heavily dependent on instrumentation sensitivity and throughput advances. The introduction of the Thermo Fisher Scientific Astral combining the Orbitrap and a novel asymmetric track lossless analyzer with optimized ion transfer and flight track design was readily awaited.30–32 The combination of 200 Hz scanning speed in the Astral analyzer with the unprecedented resolution of the Orbitrap demonstrates great potential for profiling single cells.32 This now complements SCP studies regularly performed on earlier generation Orbitrap instruments, most recently linear ion trap analyzers and the highly sensitive time-of-flight instruments with trapped ion-mobility separation for operation at 100% duty cycle.3,5,33,34 The latter has been updated this year to the Bruker Daltonics timsTOF Ultra with a more efficient ion source, allowing more ions to enter the instrument and thereby again boosting sensitivity, as well as improving dynamic range.10,35 The combination of instrument vendors striving to improve sensitivity and throughput paired with the introduction of alternative acquisition strategies and outside the box thinking is pushing the field forward every day. Our SCP initiative therefore aims to support further development, provide a comparative overview of many techniques for multidisciplinary scientists and guide study design to make SCP more readily available for the scientific community.
Contributed by Dr. Budnik Bogdan, chair of the B/D-HPP single-cell initiative
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