Author Correction: High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

Woo, Jongmin Jacob; Williams, Sarah; Markillie, Lye Meng; Feng, Song; Tsai, Chia‐Feng; Aguilera-Vazquez, Victor; Sontag, Ryan; Moore, Ronald J.; Hu, Dehong; Mehta, Hardeep S.; Cantlon-Bruce, Joshua; Liu, Tao; Adkins, Joshua N.; Smith, Richard; Clair, Gérémy; Paša‐Tolić, Ljiljana; Zhu, Ying

doi:10.1038/s41467-021-27110-0

Cited by 11 publications

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“…In contrast to the label-free analysis, the TMT-based boosting strategy can increase not only sample throughput (up to 18 channels) 48 but also detection sensitivity with the use of the carrier proteome in the boosting channel for significantly improved MS1 signal. This boosting strategy has recently been demonstrated for effective single-cell proteomics analysis 1 , 15 , 17 . It has also been applied to study low abundant PTMs such as phosphotyrosine peptides 19 or secreted glycoproteins 49 .…”

Section: Discussionmentioning

confidence: 99%

“…Sensitive and nanoscale phosphoproteomic methods have the potential to make such cell type-specific applications more feasible in clinical settings, and also enable more precise phenotypic analysis of cell (sub)types in a time-and/or dose-dependent fashion. Indeed, mass spectrometry (MS)-based single-cell proteomics has recently made significant advancements in detection sensitivity and high-throughput measurements [1][2][3][4] . A remaining technical challenge for nanoscale proteomics is the comprehensive quantitative profiling of post-translational modifications (PTMs), particularly protein phosphorylation states 5 , key indicators for signaling pathways and network activations essential for many physiological functions 6,7 .…”

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confidence: 99%

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A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

et al. 2023

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Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We develop a tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3000 (>9500) phosphopeptides from 1 (10) µg of cell lysate using the label-free method without a spectral library. It also enables precise quantification of ~600 phosphopeptides from 100 sorted cells (single-cell level input for the enriched phosphopeptides) and ~700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 µm (equivalent to ~100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.

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Section: Discussionmentioning

confidence: 99%

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A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

et al. 2023

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“… 11 , 12 , 15 The performance of NanoPOTS has improved over time in terms of versatility, sensitivity, and reproducibility by combining a nested nano-well chip with a cellenONE commercial liquid dispensing instrument. 15 The cellenONE system has also been used in proteoCHIP-based single-cell proteomics. 16 In this method, the sample solution—in the nanoliter range—is covered with hexadecane to prevent evaporation.…”

Section: Introductionmentioning

confidence: 99%

“…The nanoPOTS method uses a specially fabricated nano-well chip and a liquid handling system for digestion. These devices were designed to process the sample in a small volume to reduce protein and peptide adsorption loss. ,, The performance of NanoPOTS has improved over time in terms of versatility, sensitivity, and reproducibility by combining a nested nano-well chip with a cellenONE commercial liquid dispensing instrument . The cellenONE system has also been used in proteoCHIP-based single-cell proteomics .…”

Section: Introductionmentioning

confidence: 99%

Water Droplet-in-Oil Digestion Method for Single-Cell Proteomics

et al. 2022

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Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

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“…Sensitive and nanoscale phosphoproteomic methods have the potential to make such cell type-specific applications more feasible in clinical settings, and also enable more precise phenotypic analysis of cell (sub)types in a time-and/or dose-dependent fashion. Indeed, mass spectrometry (MS)-based single-cell proteomics has recently made significant advancements in detection sensitivity and highthroughput measurements [1][2][3][4] . A remaining technical challenge for nanoscale proteomics is the comprehensive quantitative profiling of post-translational modifications (PTMs), particularly protein phosphorylation states 5 , key indicators for signaling pathways and network activations essential for many physiological functions 6,7 .…”

Section: Introductionmentioning

confidence: 99%

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

Tsai

Wang

Hsu

et al. 2022

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Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We developed a novel tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3,000 (>9,500) phosphopeptides from 1 (10) μg of cell lysate using the label-free method without a spectral library. It also enabled precise quantification of ~600 phosphopeptides from 100 cells sorted by FACS (single-cell level input for the enriched phosphopeptides) and ~700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 μm (equivalent to ~100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.

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Author Correction: High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

Abstract: In this article the author name Chia-Feng Tsai was incorrectly written as Chai-Feng Tsai.The grant number U01 HL148860 relating to NIH grants for Joshua N. Adkins and Geremy C. Clair was omitted. The original article has been corrected.

Cited by 11 publications

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A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

Water Droplet-in-Oil Digestion Method for Single-Cell Proteomics

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

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