Recent advances in single-cell subcellular sampling

Sahota, Annie; Cabrejos, Anthony Monteza; Kwan, Zoe; Nadappuram, Binoy Paulose; Ivanov, Aleksandar P.; Edel, Joshua B.

doi:10.1039/d3cc00573a

Cited by 7 publications

(2 citation statements)

References 103 publications

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“…Currently, new developments in omics are focusing on improving sensitivity for enabling single cell omics 1 and even sub-cellular omics 2 . Technical advances in the development of mass spectrometer systems enable bottom-up proteome analysis with total protein amounts less than one ng.…”

Section: Introductionmentioning

confidence: 99%

Assay for characterizing adsorption-properties of surfaces (APS) used for sample preparation prior to quantitative omics

Siebels,

Moritz,

Hübler

et al. 2023

Preprint

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Analytes during their journey from their natural sources to their identification and quantification are prone to adsorption to surfaces before they enter an analytical instrument, causing false quantities. This problem is especially severe in diverse omics. Here, thousands of analytes with a broad range of chemical properties and thus different affinities to surfaces are quantified within a single analytical run. For quantifying adsorption effects caused by surfaces of sample handling tools, an assay was developed, applying LC-MS/MS-based differential bottom-up proteomics and as probe a reference mixture of thousands of tryptic peptides, covering a broad range of chemical properties. The assay was tested by investigating the adsorption properties of several vials composed of polypropylene, including low-protein-binding polypropylene vials, borosilicate glass vials and low-retention glass vials. In total 3531 different peptides were identified and quantified across all samples and therefore used as probes. A significant number of hydrophobic peptides adsorbed on polypropylene vials. In contrast, only very few peptides adsorbed to low-protein-binding polypropylene vials. The highest number of peptides adsorbed to glass vials, driven by electrostatic as well as hydrophobic interactions. Calculation of the impact of the adsorption of peptides on differential quantitative proteomics showed significant false results. In summary, the new assay is suitable to characterize adsorption properties of surfaces getting into contact with analytes during sample preparation, thereby giving the opportunity to find parameters for minimizing false quantities.

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Section: Introductionmentioning

confidence: 99%

Assay for characterizing adsorption-properties of surfaces (APS) used for sample preparation prior to quantitative omics

Siebels,

Moritz,

Hübler

et al. 2023

Preprint

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show abstract

“…However, all these approaches are end-point assays that require the cell to be lysed or fixed before profiling. An exciting new frontier in single cell analysis is the facilitation of temporal/sequential/longitudinal cellular profiling 3,4 . Chen, Guillaume-Gentil et al established Live-seq, a technique based on fluidic force microscopy 5 allowing the pressure-driven RNA extraction from living cells at distinct time points 6 .…”

Section: Introductionmentioning

confidence: 99%

Single Cell Track and Trace: live cell labelling and temporal transcriptomics via nanobiopsy

Marcuccio

Chau

Tanner

et al. 2023

Preprint

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Single-cell RNA sequencing has revolutionised our understanding of cellular heterogeneity, but whether using isolated cells or more recent spatial transcriptomics approaches, these methods require isolation and lysis of the cell under investigation. This provides a snapshot of the cell transcriptome from which dynamic trajectories, such as those that trigger cell state transitions, can only be inferred. Here, we present cellular nanobiopsy: a platform that enables simultaneous labelling and sampling from a single cell without killing it. The technique is based on scanning ion conductance microscopy (SICM) and uses a double-barrel nanopipette with one barrel able to inject a fluorescent dye and the other used as an electrochemical syringe to extract femtolitre-volumes of cytosol. We used the nanobiopsy to longitudinally profile the transcriptome of single glioblastoma (GBM) brain tumour cells in vitro over 72hrs with and without standard treatment. Our results suggest that treatment either induces or selects for more transcriptionally stable cells. We envision the nanobiopsy will transform standard single-cell transcriptomics from a static analysis into a dynamic and temporal assay.

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Nanoscale Methods for Longitudinal Extraction of Intracellular Contents

Quek,

Tay

2024

Advanced Materials

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Longitudinal analysis of intracellular contents including gene and protein expression is crucial for deciphering the fundamentally dynamic nature of cells. This offers invaluable insights into complex tissue composition and behavior, and drive progress in disease diagnosis, biomarker discovery and drug development. Traditional longitudinal analysis workflows, involving the destruction of cells at various timepoints, limit insights to singular moments and fail to account for cellular heterogeneity. Current non‐destructive approaches, like temporal modelling with single‐cell RNA‐sequencing and live‐cell fluorescence imaging, either rely on biological assumptions or possess risk of cellular perturbation. Recent advances in nanoscale technologies for non‐destructive intracellular content extraction offer a promising solution to these challenges. These novel methods work at the nanoscale to non‐destructively access cellular membranes and can be broadly classified into three mechanisms: tip‐facilitated aspiration, membrane‐based and probe‐based methods. This perspective focuses on these emerging nanotechnologies for repeated intracellular content extraction. We discuss their potential in longitudinal analysis, address the critical requirements for effective repeated sampling, and explore the suitability of each technique for various applications. Furthermore, we highlight unresolved challenges in repeated sampling to encourage further research in this growing field.This article is protected by copyright. All rights reserved

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Recent advances in single-cell subcellular sampling

Abstract: Recent innovations in single-cell technologies have opened up exciting possibilities for profiling the omics of individual cells. Minimally invasive analysis tools that probe and remove the contents of living cells...

Cited by 7 publications

References 103 publications

Assay for characterizing adsorption-properties of surfaces (APS) used for sample preparation prior to quantitative omics

Assay for characterizing adsorption-properties of surfaces (APS) used for sample preparation prior to quantitative omics

Single Cell Track and Trace: live cell labelling and temporal transcriptomics via nanobiopsy

Nanoscale Methods for Longitudinal Extraction of Intracellular Contents

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