Recent advances in the field of single-cell proteomics

Petrosius, Valdemaras; Schoof, Erwin M.

doi:10.1016/j.tranon.2022.101556

Cited by 47 publications

(85 citation statements)

References 107 publications

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“…To process biologically relevant samples where standard solid-phase extraction 28 cannot be used, a pre-column can be used to ensure robustness on the chromatographic system, and prevent clogging by non-protein contaminants present in the samples. This is especially relevant in single-cell proteomics 1 where indeed prior sample clean-up is not possible. With a tailor-made μ PAC pre-column setup, consisting of non-porous 5 μ m pillars based on C8, we developed 32 and 52 minute methods that could quantify similar peptide and protein group numbers as a single-column setup (Figure S4A-B).…”

Section: Resultsmentioning

confidence: 99%

“…Although invaluable, the aggregated view obtained by bulk cell population experiments is not sufficient to achieve fundamental understanding of human development and disease. The means to interrogate the first two aspects of the central dogma of biology (DNA-RNA-Protein) are well established and have been widely adopted, but the study of proteomes by liquid chromatography coupled mass spectrometry (LC-MS) at single-cell resolution is just entering the biological application phase 1 . It is estimated that a single mammalian cell contains 50-450pg of protein 2 , posing significant challenges to protein identification and quantification.…”

Section: Introductionmentioning

confidence: 99%

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Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

Petrosius

Aragon-Fernandez

Üresin

et al. 2022

Preprint

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Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, mass spectrometry-based single-cell proteomics (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carried out comprehensive analysis of orbitrap-based data independent acquisition (DIA) for limited material proteomics. Notably, we found a fundamental difference between optimal DIA methods for high- and low-load samples. We further improved our low-input DIA method by relying on high-resolution MS1 quantification, thus more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we were able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we establish a complete experimental scp-MS workflow, combining DIA with accessible single-cell sample preparation and the latest chromatographic and computational advances and showcase our developments by profiling real single cells.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

Petrosius

Aragon-Fernandez

Üresin

et al. 2022

Preprint

Self Cite

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“…As a step to circumvent such limitations, recent progress in SCP technologies, microscopy, and data analysis have started to enable the 3D mapping of proteome expression to explore single-cell heterogeneity, protein translocation events, and the network of cell-cell interactions and their spatial context. 228 To advance clinical research, SCP approaches have demonstrated the potential to map spatial distribution of proteome with subcellular resolution across tissues. 229 Many biological systems are highly heterogeneous, consisting of different cell types, intermixed subpopulations, and tissue substructures (e.g., regions with similar spatial expression patterns).…”

Section: Applications Of Ms-based Global Single-cell Proteomicsmentioning

confidence: 99%

Recent advances in microfluidics for single-cell functional proteomics

et al. 2023

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“…Protein-related difficulties include the wide range of post-translational modifications and the inability of peptides to be amplified. As a result, current efforts are primarily focused on increasingg the signal-to-noise ratio by reducing sample processing volumes and ion contamination ( 91 ). Until standardized mass spectrometry-based single-cell proteomics is available, the field is dominated by other techniques.…”

Section: Deciphering Clonal Heterogeneity and Evolution In Cllmentioning

confidence: 99%

Recent revelations and future directions using single-cell technologies in chronic lymphocytic leukemia

et al. 2023

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Chronic lymphocytic leukemia (CLL) is a clinically and biologically heterogeneous disease with varying outcomes. In the last decade, the application of next-generation sequencing technologies has allowed extensive mapping of disease-specific genomic, epigenomic, immunogenetic, and transcriptomic signatures linked to CLL pathogenesis. These technologies have improved our understanding of the impact of tumor heterogeneity and evolution on disease outcome, although they have mostly been performed on bulk preparations of nucleic acids. As a further development, new technologies have emerged in recent years that allow high-resolution mapping at the single-cell level. These include single-cell RNA sequencing for assessment of the transcriptome, both of leukemic and non-malignant cells in the tumor microenvironment; immunogenetic profiling of B and T cell receptor rearrangements; single-cell sequencing methods for investigation of methylation and chromatin accessibility across the genome; and targeted single-cell DNA sequencing for analysis of copy-number alterations and single nucleotide variants. In addition, concomitant profiling of cellular subpopulations, based on protein expression, can also be obtained by various antibody-based approaches. In this review, we discuss different single-cell sequencing technologies and how they have been applied so far to study CLL onset and progression, also in response to treatment. This latter aspect is particularly relevant considering that we are moving away from chemoimmunotherapy to targeted therapies, with a potentially distinct impact on clonal dynamics. We also discuss new possibilities, such as integrative multi-omics analysis, as well as inherent limitations of the different single-cell technologies, from sample preparation to data interpretation using available bioinformatic pipelines. Finally, we discuss future directions in this rapidly evolving field.

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Recent advances in the field of single-cell proteomics

Cited by 47 publications

References 107 publications

Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

Recent advances in microfluidics for single-cell functional proteomics

Recent revelations and future directions using single-cell technologies in chronic lymphocytic leukemia

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