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Proteomic Workflows for High-Quality Quantitative Proteome and Post-Translational Modification Analysis of Clinically Relevant Samples from Formalin-Fixed Paraffin-Embedded Archives

Kuras, Magdalena LU orcid ; Woldmar, Nicole LU ; Kim, Yonghyo LU ; Hefner, Max LU ; Malm, Johan LU ; Moldvay, Judit ; Dome, Balazs LU ; Fillinger, Janos ; Pizzatti, Luciana and Gil, Jeovanis LU , et al. (2021) In Journal of Proteome Research 20(1). p.1027-1039
Abstract

Well-characterized archival formalin-fixed paraffin-embedded (FFPE) tissues are of much value for prospective biomarker discovery studies, and protocols that offer high throughput and good reproducibility are essential in proteomics. Therefore, we implemented efficient paraffin removal and protein extraction from FFPE tissues followed by an optimized two-enzyme digestion using suspension trapping (S-Trap). The protocol was then combined with TMTpro 16plex labeling and applied to lung adenocarcinoma patient samples. In total, 9585 proteins were identified, and proteins related to the clinical outcome were detected. Because acetylation is known to play a major role in cancer development, a fast on-trap acetylation protocol was developed... (More)

Well-characterized archival formalin-fixed paraffin-embedded (FFPE) tissues are of much value for prospective biomarker discovery studies, and protocols that offer high throughput and good reproducibility are essential in proteomics. Therefore, we implemented efficient paraffin removal and protein extraction from FFPE tissues followed by an optimized two-enzyme digestion using suspension trapping (S-Trap). The protocol was then combined with TMTpro 16plex labeling and applied to lung adenocarcinoma patient samples. In total, 9585 proteins were identified, and proteins related to the clinical outcome were detected. Because acetylation is known to play a major role in cancer development, a fast on-trap acetylation protocol was developed for studying endogenous lysine acetylation, which allows identification and localization of the lysine acetylation together with quantitative comparison between samples. We demonstrated that FFPE tissues are equivalent to frozen tissues to study the degree of acetylation between patients. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that resolves known proteomic-related challenges. We demonstrate compatibility of the S-Trap with isobaric labeling and for the first time, we prove that it is feasible to study endogenous lysine acetylation stoichiometry in FFPE tissues, contributing to better utility of the existing global tissue archives. The MS proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020157, PXD021986, and PXD021964.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
cancer, clinical proteomics, FFPE tissue, high throughput, isobaric labeling (TMT), lung adenocarcinoma, lysine acetylation stoichiometry, mass spectrometry, sample preparation, suspension trapping (S-Trap)
in
Journal of Proteome Research
volume
20
issue
1
pages
1027 - 1039
publisher
The American Chemical Society (ACS)
external identifiers
  • pmid:33301673
  • scopus:85098765624
ISSN
1535-3893
DOI
10.1021/acs.jproteome.0c00850
language
English
LU publication?
yes
id
c08a1498-4799-44d2-a605-13a3ffbf237b
date added to LUP
2021-01-13 11:56:43
date last changed
2024-04-18 00:35:25
@article{c08a1498-4799-44d2-a605-13a3ffbf237b,
  abstract     = {{<p>Well-characterized archival formalin-fixed paraffin-embedded (FFPE) tissues are of much value for prospective biomarker discovery studies, and protocols that offer high throughput and good reproducibility are essential in proteomics. Therefore, we implemented efficient paraffin removal and protein extraction from FFPE tissues followed by an optimized two-enzyme digestion using suspension trapping (S-Trap). The protocol was then combined with TMTpro 16plex labeling and applied to lung adenocarcinoma patient samples. In total, 9585 proteins were identified, and proteins related to the clinical outcome were detected. Because acetylation is known to play a major role in cancer development, a fast on-trap acetylation protocol was developed for studying endogenous lysine acetylation, which allows identification and localization of the lysine acetylation together with quantitative comparison between samples. We demonstrated that FFPE tissues are equivalent to frozen tissues to study the degree of acetylation between patients. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that resolves known proteomic-related challenges. We demonstrate compatibility of the S-Trap with isobaric labeling and for the first time, we prove that it is feasible to study endogenous lysine acetylation stoichiometry in FFPE tissues, contributing to better utility of the existing global tissue archives. The MS proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020157, PXD021986, and PXD021964.</p>}},
  author       = {{Kuras, Magdalena and Woldmar, Nicole and Kim, Yonghyo and Hefner, Max and Malm, Johan and Moldvay, Judit and Dome, Balazs and Fillinger, Janos and Pizzatti, Luciana and Gil, Jeovanis and Marko-Varga, Gyorgy and Rezeli, Melinda}},
  issn         = {{1535-3893}},
  keywords     = {{cancer; clinical proteomics; FFPE tissue; high throughput; isobaric labeling (TMT); lung adenocarcinoma; lysine acetylation stoichiometry; mass spectrometry; sample preparation; suspension trapping (S-Trap)}},
  language     = {{eng}},
  number       = {{1}},
  pages        = {{1027--1039}},
  publisher    = {{The American Chemical Society (ACS)}},
  series       = {{Journal of Proteome Research}},
  title        = {{Proteomic Workflows for High-Quality Quantitative Proteome and Post-Translational Modification Analysis of Clinically Relevant Samples from Formalin-Fixed Paraffin-Embedded Archives}},
  url          = {{http://dx.doi.org/10.1021/acs.jproteome.0c00850}},
  doi          = {{10.1021/acs.jproteome.0c00850}},
  volume       = {{20}},
  year         = {{2021}},
}