On-capillary alkylation micro-reactor: a facile strategy for proteo-metabolome profiling in the same single cells

He, Yingyun; Yuan, Huiming; Liang, Yu; Liu, Xinxin; Zhang, Xiaozhe; Ji, Yahui; Zhao, Baofeng; Yang, Kaiguang; Zhang, Jue; Zhang, Shen; Zhang, Yukui; Zhang, Lihua

doi:10.1039/d3sc05047e

Cited by 8 publications

(3 citation statements)

References 47 publications

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“…37 Recently, Zhang's group performed the preseparation of proteins and metabolites by using an oncapillary alkylation microreactor before the MS detection for single mouse oocyte proteome and metabolome analysis, with a total of 3457 proteins and 171 metabolites identified in single oocytes. 38 Despite the success of the above-mentioned singlecell proteomic and metabolomic profiling approaches, simple and easy-to-use dual-omic methods for proteomic and metabolomic analysis using LC-MS systems as for routine bulk samples are still urgently needed.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Zhao et al placed a single cell in a 10 pL scale microwell chip and used an automated picoliter extraction system to sequentially sample metabolites, lipids, and proteins of the cell for pulse-ESI-MS analysis with 1391 proteins, phospholipids, and metabolites detected from one single cell . Recently, Zhang’s group performed the preseparation of proteins and metabolites by using an on-capillary alkylation microreactor before the MS detection for single mouse oocyte proteome and metabolome analysis, with a total of 3457 proteins and 171 metabolites identified in single oocytes . Despite the success of the above-mentioned single-cell proteomic and metabolomic profiling approaches, simple and easy-to-use dual-omic methods for proteomic and metabolomic analysis using LC-MS systems as for routine bulk samples are still urgently needed.…”

Section: Introductionmentioning

confidence: 99%

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One-Shot Single-Cell Proteome and Metabolome Analysis Strategy for the Same Single Cell

Wu,

Xu,

Jiang

et al. 2024

Anal. Chem.

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Characterizing the profiles of proteome and metabolome at the single-cell level is of great significance in single-cell multiomic studies. Herein, we proposed a novel strategy called one-shot single-cell proteome and metabolome analysis (scPMA) to acquire the proteome and metabolome information in a single-cell individual in one injection of LC-MS/MS analysis. Based on the scPMA strategy, a total workflow was developed to achieve the single-cell capture, nanoliter-scale sample pretreatment, one-shot LC injection and separation of the enzyme-digested peptides and metabolites, and dual-zone MS/MS detection for proteome and metabolome profiling. Benefiting from the scPMA strategy, we realized dual-omic analysis of single tumor cells, including A549, HeLa, and HepG2 cells with 816, 578, and 293 protein groups and 72, 91, and 148 metabolites quantified on average. A single-cell perspective experiment for investigating the doxorubicin-induced antitumor effects in both the proteome and metabolome aspects was also performed.

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Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

One-Shot Single-Cell Proteome and Metabolome Analysis Strategy for the Same Single Cell

Wu,

Xu,

Jiang

et al. 2024

Anal. Chem.

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“…Key to improving this process is handling samples in small volumes, which effectively reduces analyte dilution and minimizes interaction with adsorptive surfaces, thus benefiting single-cell proteomics . Preventing sample loss during preparation is critical, prompting the development of ultralow volume sample processing techniques, such as OAD, nanoPOTS, , iPAD-1, OCAM, and certain multiwell plate methods, ,− which have shown promising results.…”

Section: Introductionmentioning

confidence: 99%

AM-DMF-SCP: Integrated Single-Cell Proteomics Analysis on an Active Matrix Digital Microfluidic Chip

Yang,

Jin,

Chen

et al. 2024

JACS Au

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Single-cell proteomics offers unparalleled insights into cellular diversity and molecular mechanisms, enabling a deeper understanding of complex biological processes at the individual cell level. Here, we develop an integrated sample processing on an activematrix digital microfluidic chip for single-cell proteomics (AM-DMF-SCP). Employing the AM-DMF-SCP approach and data-independent acquisition (DIA), we identify an average of 2258 protein groups in single HeLa cells within 15 min of the liquid chromatography gradient. We performed comparative analyses of three tumor cell lines: HeLa, A549, and HepG2, and machine learning was utilized to identify the unique features of these cell lines. Applying the AM-DMF-SCP to characterize the proteomes of a third-generation EGFR inhibitor, ASK120067-resistant cells (67R) and their parental NCI-H1975 cells, we observed a potential correlation between elevated VIM expression and 67R resistance, which is consistent with the findings from bulk sample analyses. These results suggest that AM-DMF-SCP is an automated, robust, and sensitive platform for single-cell proteomics and demonstrate the potential for providing valuable insights into cellular mechanisms.

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Single‐Cell Secretome Profiling Enabled by Selective Enrichment and NanoLC‐MS/MS Analysis

He,

Liu,

Zhu

et al. 2024

Angewandte Chemie

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Recent advances in single‐cell proteomics enable the direct profiling of thousands of proteins from a single mammalian cell. However, due to the bottlenecks in detecting low‐abundance secreted proteins and extracellular vesicle (EV) proteins (collectively referred to as the secretome) against a background of high‐abundance proteins in serum‐containing culture medium, the comprehensive investigation of the secretome at the single‐cell level using nanoLC‐MS/MS still remains challenging. Herein, we report a novel single‐cell secretome profiling (SCSP) method by integrating the metabolic labeling of newly synthesized proteins, click chemistry‐based enrichment, and in situ digestion of the labeled secretome in an alkyne‐functionalized capillary micro‐reactor, followed by nanoLC‐MS/MS analysis. By this method, an average of 389 protein groups were quantified from the secretome of single HeLa cells (n = 17), with a total of 752 protein groups confidently identified in the single‐cell secretome, which is a significant increase compared to the previously reported targeted analysis limited to dozens of secreted proteins by antibody recognition. These results indicated that our developed SCSP method would provide a powerful tool to gain insights into secretion heterogeneity and intercellular communication at the single‐cell level.

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On-capillary alkylation micro-reactor: a facile strategy for proteo-metabolome profiling in the same single cells

Abstract: An on-capillary alkylation micro-reactor (OCAM) was developed for parallel measurement of proteome and metabolome in the same single cells, providing a new avenue for studying system biology at single-cell level.

Cited by 8 publications

References 47 publications

One-Shot Single-Cell Proteome and Metabolome Analysis Strategy for the Same Single Cell

One-Shot Single-Cell Proteome and Metabolome Analysis Strategy for the Same Single Cell

AM-DMF-SCP: Integrated Single-Cell Proteomics Analysis on an Active Matrix Digital Microfluidic Chip

Single‐Cell Secretome Profiling Enabled by Selective Enrichment and NanoLC‐MS/MS Analysis

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