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Quantitative Proteomic Analysis of Microdissected Breast Cancer Tissues: Comparison of Label-Free and SILAC-based Quantification with Shotgun, Directed, and Targeted MS Approaches
journal contribution
posted on 2016-02-18, 18:23 authored by 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 UmarQuantitative
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.