Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome

Chaewon Kang; Sunghyun Huh; Dowoon Nam; Hokeun Kim; Jiwon Hong; Daehee Hwang; Sang Won Lee

doi:10.1021/acs.analchem.2c02641

Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome

Chaewon Kang, Sunghyun Huh, Dowoon Nam, Hokeun Kim, Jiwon Hong, Daehee Hwang, Sang Won Lee

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

Abstract

Protein phosphorylation is a prevalent post-translational modification that regulates essentially every aspect of cellular processes. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive offline sample fractionation and a phosphopeptide enrichment method is a best practice for deep phosphoproteome profiling, but balancing throughput and profiling depth remains a practical challenge. We present an online three-dimensional separation method for ultradeep phosphoproteome profiling that combines an online two-dimensional liquid chromatography separation and an additional gas-phase separation. This method identified over 100,000 phosphopeptides (>60,000 phosphosites) in HeLa cells during 1.5 days of data acquisition, and the largest HeLa cell phosphoproteome significantly expanded the detectable functional landscape of cellular phosphoproteome.

Original language	English
Pages (from-to)	12185-12195
Number of pages	11
Journal	Analytical chemistry
Volume	94
Issue number	35
DOIs	https://doi.org/10.1021/acs.analchem.2c02641
Publication status	Published - 2022 Sept 6

Bibliographical note

Funding Information:
This work was supported by the Korea Basic Science Institute (KBSI) National Research Facilities & Equipment Center (NFEC) funded by the Korea government (Ministry of Education) (2019R1A6C1010028).

Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.

ASJC Scopus subject areas

Analytical Chemistry

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10.1021/acs.analchem.2c02641

Cite this

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title = "Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome",

abstract = "Protein phosphorylation is a prevalent post-translational modification that regulates essentially every aspect of cellular processes. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive offline sample fractionation and a phosphopeptide enrichment method is a best practice for deep phosphoproteome profiling, but balancing throughput and profiling depth remains a practical challenge. We present an online three-dimensional separation method for ultradeep phosphoproteome profiling that combines an online two-dimensional liquid chromatography separation and an additional gas-phase separation. This method identified over 100,000 phosphopeptides (>60,000 phosphosites) in HeLa cells during 1.5 days of data acquisition, and the largest HeLa cell phosphoproteome significantly expanded the detectable functional landscape of cellular phosphoproteome.",

author = "Chaewon Kang and Sunghyun Huh and Dowoon Nam and Hokeun Kim and Jiwon Hong and Daehee Hwang and Lee, {Sang Won}",

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doi = "10.1021/acs.analchem.2c02641",

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AU - Huh, Sunghyun

AU - Nam, Dowoon

AU - Kim, Hokeun

AU - Hong, Jiwon

AU - Hwang, Daehee

AU - Lee, Sang Won

N1 - Funding Information: This work was supported by the Korea Basic Science Institute (KBSI) National Research Facilities & Equipment Center (NFEC) funded by the Korea government (Ministry of Education) (2019R1A6C1010028). Publisher Copyright: © 2022 American Chemical Society. All rights reserved.

PY - 2022/9/6

Y1 - 2022/9/6

N2 - Protein phosphorylation is a prevalent post-translational modification that regulates essentially every aspect of cellular processes. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive offline sample fractionation and a phosphopeptide enrichment method is a best practice for deep phosphoproteome profiling, but balancing throughput and profiling depth remains a practical challenge. We present an online three-dimensional separation method for ultradeep phosphoproteome profiling that combines an online two-dimensional liquid chromatography separation and an additional gas-phase separation. This method identified over 100,000 phosphopeptides (>60,000 phosphosites) in HeLa cells during 1.5 days of data acquisition, and the largest HeLa cell phosphoproteome significantly expanded the detectable functional landscape of cellular phosphoproteome.

AB - Protein phosphorylation is a prevalent post-translational modification that regulates essentially every aspect of cellular processes. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive offline sample fractionation and a phosphopeptide enrichment method is a best practice for deep phosphoproteome profiling, but balancing throughput and profiling depth remains a practical challenge. We present an online three-dimensional separation method for ultradeep phosphoproteome profiling that combines an online two-dimensional liquid chromatography separation and an additional gas-phase separation. This method identified over 100,000 phosphopeptides (>60,000 phosphosites) in HeLa cells during 1.5 days of data acquisition, and the largest HeLa cell phosphoproteome significantly expanded the detectable functional landscape of cellular phosphoproteome.

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Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome

Abstract

Bibliographical note

ASJC Scopus subject areas

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