Advanced

Quantitative proteomic analysis of microdissected breast cancer tissues : comparison of label-free and SILAC-based quantification with shotgun, directed, and targeted MS approaches

Liu, Ning Qing; Dekker, Lennard J M; Stingl, Christoph; Güzel, Coşkun; De Marchi, Tommaso LU ; Martens, John W. M.; Foekens, John A.; Luider, Theo M. and Umar, Arzu (2013) In Journal of Proteome Research 12(10). p.41-4627
Abstract

Quantitative proteomics plays an important role in validation of breast-cancer-related biomarkers. In this study, we systematically compared the performance of label-free quantification (LFQ) and SILAC with shotgun and directed methods for quantifying breast-cancer-related markers in microdissected tissues. We show that LFQ leads to slightly higher coefficient of variation (CV) for protein quantification (median CV = 16.3%) than SILAC quantification (median CV = 13.7%) (P < 0.0001), but LFQ method enables ∼60% more protein quantification and is also more reproducible (∼20% more proteins were quantified in all replicate samples). Furthermore, we describe a method to accurately quantify multiple proteins within one pathway, that is,... (More)

Quantitative proteomics plays an important role in validation of breast-cancer-related biomarkers. In this study, we systematically compared the performance of label-free quantification (LFQ) and SILAC with shotgun and directed methods for quantifying breast-cancer-related markers in microdissected tissues. We show that LFQ leads to slightly higher coefficient of variation (CV) for protein quantification (median CV = 16.3%) than SILAC quantification (median CV = 13.7%) (P < 0.0001), but LFQ method enables ∼60% more protein quantification and is also more reproducible (∼20% more proteins were quantified in all replicate samples). Furthermore, we describe a method to accurately quantify multiple proteins within one pathway, that is, "focal adhesion pathway", in trace amounts of breast cancer tissues using a SILAC-based SRM assay. Using this SILAC-based SRM assay, we precisely quantified five "focal adhesion" proteins with good quantitative precision (CV range: 2.4-5.9%) in replicate whole tissue lysate samples and replicate microdissected samples (CV range: 5.8-16.1%). Our results show that in microdissected breast cancer tissues LFQ in combination with shotgun proteomics performed the best overall and is therefore suitable for both biomarker discovery and validation in these types of specimens. The SILAC-based SRM method can be used for the development of clinically relevant protein assays in tumor biopsies.

(Less)
Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
keywords
Amino Acid Sequence, Biomarkers, Tumor, Breast Neoplasms, Cell Adhesion Molecules, Cell Line, Tumor, Female, Focal Adhesions, Humans, Isotope Labeling, Laser Capture Microdissection, Molecular Sequence Data, Proteome, Proteomics, Reference Standards, Reproducibility of Results, Tandem Mass Spectrometry, Comparative Study, Journal Article, Research Support, Non-U.S. Gov't
in
Journal of Proteome Research
volume
12
issue
10
pages
41 - 4627
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:84885234215
ISSN
1535-3893
DOI
10.1021/pr4005794
language
English
LU publication?
no
id
d633035b-5f4c-4264-8b7f-9f5d15a2c564
date added to LUP
2017-06-27 14:27:06
date last changed
2019-07-09 04:04:36
@article{d633035b-5f4c-4264-8b7f-9f5d15a2c564,
  abstract     = {<p>Quantitative proteomics plays an important role in validation of breast-cancer-related biomarkers. In this study, we systematically compared the performance of label-free quantification (LFQ) and SILAC with shotgun and directed methods for quantifying breast-cancer-related markers in microdissected tissues. We show that LFQ leads to slightly higher coefficient of variation (CV) for protein quantification (median CV = 16.3%) than SILAC quantification (median CV = 13.7%) (P &lt; 0.0001), but LFQ method enables ∼60% more protein quantification and is also more reproducible (∼20% more proteins were quantified in all replicate samples). Furthermore, we describe a method to accurately quantify multiple proteins within one pathway, that is, "focal adhesion pathway", in trace amounts of breast cancer tissues using a SILAC-based SRM assay. Using this SILAC-based SRM assay, we precisely quantified five "focal adhesion" proteins with good quantitative precision (CV range: 2.4-5.9%) in replicate whole tissue lysate samples and replicate microdissected samples (CV range: 5.8-16.1%). Our results show that in microdissected breast cancer tissues LFQ in combination with shotgun proteomics performed the best overall and is therefore suitable for both biomarker discovery and validation in these types of specimens. The SILAC-based SRM method can be used for the development of clinically relevant protein assays in tumor biopsies.</p>},
  author       = {Liu, Ning Qing and Dekker, Lennard J M and Stingl, Christoph and Güzel, Coşkun and De Marchi, Tommaso and Martens, John W. M. and Foekens, John A. and Luider, Theo M. and Umar, Arzu},
  issn         = {1535-3893},
  keyword      = {Amino Acid Sequence,Biomarkers, Tumor,Breast Neoplasms,Cell Adhesion Molecules,Cell Line, Tumor,Female,Focal Adhesions,Humans,Isotope Labeling,Laser Capture Microdissection,Molecular Sequence Data,Proteome,Proteomics,Reference Standards,Reproducibility of Results,Tandem Mass Spectrometry,Comparative Study,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  month        = {10},
  number       = {10},
  pages        = {41--4627},
  publisher    = {The American Chemical Society (ACS)},
  series       = {Journal of Proteome Research},
  title        = {Quantitative proteomic analysis of microdissected breast cancer tissues : comparison of label-free and SILAC-based quantification with shotgun, directed, and targeted MS approaches},
  url          = {http://dx.doi.org/10.1021/pr4005794},
  volume       = {12},
  year         = {2013},
}