SCoPE2 Protocol : "Multiplexed single-cell proteomics with SCoPE2"
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Nikolai Slavov
PROVIDER: MSV000087041 | MassIVE | Fri Mar 12 08:36:00 GMT 2021
REPOSITORIES: MassIVE
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Project description:Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.
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Project description:Single-cell proteomics workflows have considerably improved in sensitivity and reproducibility to characterize as-yet unknown biological phenomena. With the emergence of multiplexed single-cell proteomics, studies increasingly present single-cell measurements in conjunction with an abundant congruent carrier to improve the precursor selection and enhance identifications. While these extreme carrier spikes are often >100× more abundant than the investigated samples, the total ion current undoubtably increases but the quantitative accuracy possibly is affected. We here focus on narrowly titrated carrier spikes (i.e., <20×) and assess their elimination for a comparable sensitivity with superior accuracy. We find that subtle changes in the carrier ratio can severely impact the measurement variability and describe alternative multiplexing strategies to evaluate data quality. Lastly, we demonstrate elevated replicate overlap while preserving acquisition throughput at an improved quantitative accuracy with DIA-TMT and discuss optimized experimental designs for multiplexed proteomics of trace samples. This comprehensive benchmarking gives an overview of currently available techniques and guides the conceptualization of the optimal single-cell proteomics experiment.
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Project description:BackgroundMacrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quantitative single-cell protein analysis.ResultsTo overcome this limitation, we develop SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantifies over 3042 proteins in 1490 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. Parallel measurements of transcripts by 10× Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus, SCoPE2 supports quantification with improved count statistics. This allowed exploring regulatory interactions, such as interactions between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53.ConclusionsEven in a homogeneous environment, macrophage proteomes are heterogeneous. This heterogeneity correlates to the inflammatory axis of classically and alternatively activated macrophages. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.
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2020-10-23 | GSE153673 | GEO
Project description:In this work, we compared different protocols to prepare single-cell suspensions used for scRNAseq and suggest an optimized dissociation protocol for mouse retina, which preserves cell morphology to a higher level leading to an overall increase of gene number per cell. We compared scRNAseq libraries generated with our optimized protocol to publicly available scRNAseq data of mouse retina. We further demonstrate a pipeline to reduce noise in scRNAseq caused by multiplets and ambient RNA.
2020-10-23 | GSE153672 | GEO
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Project description:The fate and physiology of individual cells are controlled by proteins. Yet, our ability to quantitatively analyze proteins in single cells has remained limited. To overcome this barrier, we developed SCoPE2. It substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enabled us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiated into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantified over 3,042 proteins in 1,490 single monocytes and macrophages in ten days of instrument time, and the quantified proteins allowed us to discern single cells by cell type. Furthermore, the data uncovered a continuous gradient of proteome states for the macrophage-like cells, suggesting that macrophage heterogeneity may emerge even in the absence of polarizing cytokines. Parallel measurements of transcripts by 10x Genomics scRNA-seq suggest that our measurements sampled 20-fold more protein copies than RNA copies per gene, and thus SCoPE2 supports quantification with improved count statistics. Joint analysis of the data illustrates how variability across single cells can reveal transcriptional and post-transcriptional gene regulation. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass-spectrometry.
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