Protocol for high-throughput semi-automated label-free- or TMT-based phosphoproteome profiling

Claire Koenig; Ana Martinez-Val; Previn Naicker; Stoyan Stoychev; Justin Jordaan; Jesper V. Olsen

doi:10.1016/j.xpro.2023.102536

Protocol for high-throughput semi-automated label-free- or TMT-based phosphoproteome profiling

Claire Koenig, Ana Martinez-Val^*, Previn Naicker, Stoyan Stoychev, Justin Jordaan, Jesper V. Olsen

^*Corresponding author for this work

Research output: Contribution to journal › Journal article › Research › peer-review

7 Citations (Scopus)

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Abstract

Tandem mass tags data-dependent acquisition (TMT-DDA) as well as data-independent acquisition-based label-free quantification (LFQ-DIA) have become the leading workflows to achieve deep proteome and phosphoproteome profiles. We present a modular pipeline for TMT-DDA and LFQ-DIA that integrates steps to perform scalable phosphoproteome profiling, including protein lysate extraction, clean-up, digestion, phosphopeptide enrichment, and TMT-labeling. We also detail peptide and/or phosphopeptide fractionation and pre-mass spectrometry desalting and provide researchers guidance on choosing the best workflow based on sample number and input. For complete details on the use and execution of this protocol, please refer to Koenig et al.¹ and Martínez-Val et al.²

Original language	English
Article number	102536
Journal	STAR Protocols
Volume	4
Issue number	3
Number of pages	30
ISSN	2666-1667
DOIs	https://doi.org/10.1016/j.xpro.2023.102536
Publication status	Published - 2023

Bibliographical note

Publisher Copyright:
© 2023 The Authors

Keywords

Mass Spectrometry
Protein Biochemistry
Proteomics

Access to Document

10.1016/j.xpro.2023.102536Licence: CC BY-NC-ND

FulltextFinal published version, 8.28 MBLicence: CC BY-NC-ND

Cite this

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title = "Protocol for high-throughput semi-automated label-free- or TMT-based phosphoproteome profiling",

abstract = "Tandem mass tags data-dependent acquisition (TMT-DDA) as well as data-independent acquisition-based label-free quantification (LFQ-DIA) have become the leading workflows to achieve deep proteome and phosphoproteome profiles. We present a modular pipeline for TMT-DDA and LFQ-DIA that integrates steps to perform scalable phosphoproteome profiling, including protein lysate extraction, clean-up, digestion, phosphopeptide enrichment, and TMT-labeling. We also detail peptide and/or phosphopeptide fractionation and pre-mass spectrometry desalting and provide researchers guidance on choosing the best workflow based on sample number and input. For complete details on the use and execution of this protocol, please refer to Koenig et al.1 and Mart{\'i}nez-Val et al.2",

keywords = "Mass Spectrometry, Protein Biochemistry, Proteomics",

author = "Claire Koenig and Ana Martinez-Val and Previn Naicker and Stoyan Stoychev and Justin Jordaan and Olsen, {Jesper V.}",

note = "Publisher Copyright: {\textcopyright} 2023 The Authors",

year = "2023",

doi = "10.1016/j.xpro.2023.102536",

language = "English",

volume = "4",

journal = "STAR Protocols",

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TY - JOUR

T1 - Protocol for high-throughput semi-automated label-free- or TMT-based phosphoproteome profiling

AU - Koenig, Claire

AU - Martinez-Val, Ana

AU - Naicker, Previn

AU - Stoychev, Stoyan

AU - Jordaan, Justin

AU - Olsen, Jesper V.

PY - 2023

Y1 - 2023

N2 - Tandem mass tags data-dependent acquisition (TMT-DDA) as well as data-independent acquisition-based label-free quantification (LFQ-DIA) have become the leading workflows to achieve deep proteome and phosphoproteome profiles. We present a modular pipeline for TMT-DDA and LFQ-DIA that integrates steps to perform scalable phosphoproteome profiling, including protein lysate extraction, clean-up, digestion, phosphopeptide enrichment, and TMT-labeling. We also detail peptide and/or phosphopeptide fractionation and pre-mass spectrometry desalting and provide researchers guidance on choosing the best workflow based on sample number and input. For complete details on the use and execution of this protocol, please refer to Koenig et al.1 and Martínez-Val et al.2

AB - Tandem mass tags data-dependent acquisition (TMT-DDA) as well as data-independent acquisition-based label-free quantification (LFQ-DIA) have become the leading workflows to achieve deep proteome and phosphoproteome profiles. We present a modular pipeline for TMT-DDA and LFQ-DIA that integrates steps to perform scalable phosphoproteome profiling, including protein lysate extraction, clean-up, digestion, phosphopeptide enrichment, and TMT-labeling. We also detail peptide and/or phosphopeptide fractionation and pre-mass spectrometry desalting and provide researchers guidance on choosing the best workflow based on sample number and input. For complete details on the use and execution of this protocol, please refer to Koenig et al.1 and Martínez-Val et al.2

KW - Mass Spectrometry

KW - Protein Biochemistry

KW - Proteomics

U2 - 10.1016/j.xpro.2023.102536

DO - 10.1016/j.xpro.2023.102536

M3 - Journal article

C2 - 37659085

AN - SCOPUS:85169561202

SN - 2666-1667

VL - 4

JO - STAR Protocols

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ER -