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