Quantitative Proteomic Analysis of Microdissected
Breast Cancer Tissues: Comparison of Label-Free and SILAC-based Quantification
with Shotgun, Directed, and Targeted MS Approaches
Ning Qing Liu
Lennard J. M. Dekker
Christoph Stingl
Coşkun Güzel
Tommaso De Marchi
John W. M. Martens
John A. Foekens
Theo M. Luider
Arzu Umar
10.1021/pr4005794.s010
https://acs.figshare.com/articles/dataset/Quantitative_Proteomic_Analysis_of_Microdissected_Breast_Cancer_Tissues_Comparison_of_Label_Free_and_SILAC_based_Quantification_with_Shotgun_Directed_and_Targeted_MS_Approaches/2370778
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%) (<i>P</i> < 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.
2016-02-18 18:23:41
Microdissected Breast Cancer Tissues
assay
SILAC
method
CV range
SRM
breast cancer tissues
tissue lysate samples
microdissected breast cancer tissues LFQ
protein quantification
Targeted MS ApproachesQuantitative proteomics
Quantitative Proteomic Analysis