Dataset Information

Easy and Accessible Workflow for Label-Free Single-Cell Proteomics

ABSTRACT: Single cell proteomics (SCP) can provide information that is unattainable through either bulk-scale protein measurements or single-cell profiling of other omes. Maximizing proteome coverage often requires custom instrumentation, consumables and reagents for sample processing and separations, which limits accessibility of SCP to a small number of specialized laboratories. Commercial platforms have become available for SCP cell isolation and sample preparation, but the high cost of these platforms and the technical expertise required for their operation place them out of reach of many interested laboratories. Here, we assessed the new HP D100 Single Cell Dispenser for label-free SCP. The low-cost instrument proved highly accurate and reproducible for dispensing reagents in the 200 nL to 2 µL range. We used the HP D100 to isolate and prepare single cells for SCP within 384-well PCR plates. When the well plates were immediately centrifuged following cell dispensing and again after reagent dispensing, we found that ~97% of wells that were identified in the instrument software as containing a single cell indeed provided proteome coverage expected of a single cell. The D100 Single Cell Dispenser combined with one-step sample processing provides a very rapid easy-to-use workflow for SCP with no reduction in proteome coverage relative to a nanowell-based workflow. The commercial well plates also facilitate autosampling with commercial instrumentation. Single cell samples were analyzed using home-packed 30-µm-i.d. nanoLC columns as well as commercially available 50-µm-i.d. columns. The commercial columns led to identification of ~35% fewer identified proteins. However, combined with the well plate-based preparation platform the presented workflow provides fully commercial and relatively low cost alternative for SCP sample preparations and separations, which should greatly broaden accessibility of SCP to other laboratories.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Jurkat Cell, Hela Cell

SUBMITTER: Ryan Kelly

LAB HEAD: Ryan T. Kelly

PROVIDER: PXD043355 | Pride | 2023-10-09

REPOSITORIES: Pride

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Easy and Accessible Workflow for Label-Free Single-Cell Proteomics.

Sanchez-Avila Ximena X Truong Thy T Xie Xiaofeng X Webber Kei G I KGI Johnston S Madisyn SM Lin Hsien-Jung L HL Axtell Nathaniel B NB Puig-Sanvicens Veronica V Kelly Ryan T RT

Journal of the American Society for Mass Spectrometry 20230818 10

Single-cell proteomics (SCP) can provide information that is unattainable through either bulk-scale protein measurements or single-cell profiling of other omes. Maximizing proteome coverage often requires custom instrumentation, consumables, and reagents for sample processing and separations, which has limited the accessibility of SCP to a small number of specialized laboratories. Commercial platforms have become available for SCP cell isolation and sample preparation, but the high cost of these ...[more]

PMID: 37594399

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Project description:The sensitivity of single-cell proteomics (SCP) has increased dramatically in recent years due to advances in experimental design, sample preparation, separations and mass spectrometry instrumentation. However, further increasing the sensitivity of SCP methods and instrumentation will enable the study of proteins within single cells that are expressed at copy numbers too small to be measured by current methods. Here we further increase SCP sensitivity by combining efficient nanoPOTS sample preparation and ultra-low-flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to extend the achievable proteome coverage for single cells to >3,000 for single-cell data analyses. WWA is based on data-dependent acquisition (DDA) but employs larger precursor isolation windows to intentionally co-isolate and co-fragment additional precursors along with the selected precursor. The resulting chimeric MS2 spectra are then resolved using the CHIMERYS search engine within Proteome Discoverer 3.0. Compared to standard DDA workflows, WWA employing isolation windows of 8-12 Th increases peptide and proteome coverage by ~28% and ~39%, respectively. For a 40 min LC gradient operated at ~15 nL/min, we identified an average of 2,150 proteins per single-cell-sized aliquots of protein digest directly from MS2 spectra, which increased to an average 3,524 proteins including proteins identified with MS1-level feature matching. Reducing the active gradient to 20 min resulted in only a 10% decrease in proteome coverage. We also compared performance of WWA with DIA underperformed relative to WWA in terms of proteome coverage, especially with faster separations. Average proteome coverage for single HeLa and K562 cells was respectively 1,758 and 1,642 based on MS2 identifications with 1% false discovery rate and 3042 and 2891 with MS1 feature matching. As such, WWA combined with efficient sample preparation and rapid separations extends the depths of the proteome that can be studied at the single-cell level.

Project description:The analysis of single cell proteomes has recently become a viable complement to transcript and genomics studies. Proteins are the main driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Both multiplexed and label-free mass spectrometry-based approaches with single cell resolution have lately attributed surprising heterogeneity to believed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lacks behind. Here, we introduce the proteoCHIP, a universal option for single cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single cell isolation and picoliter dispensing, the cellenONE®, allows to reduce final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error prone manual sample handling and overcoming evaporation. With this specialized workflow we achieved around 1,000 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,000 protein groups across 158 multiplexed single cells from two highly similar human cell types and clustered them based on their proteome. In-depth investigation of regulated proteins readily identified one of the main drivers for tumorigenicity in this cell type. Our workflow is compatible with all labeling reagents, can be easily adapted to custom workflows and is a viable option for label-free sample preparation. The specialized proteoCHIP design allows for the direct injection of label-free single cells via a standard autosampler resulting in the recovery of 30% more protein groups compared to samples transferred to PEG coated vials. We therefore are confident that our versatile, sensitive, and automated sample preparation workflow will be easily adoptable by non-specialized groups and will drive biological applications of single cell proteomics.

Dataset Information

Easy and Accessible Workflow for Label-Free Single-Cell Proteomics

Publications

Easy and Accessible Workflow for Label-Free Single-Cell Proteomics.

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