Quantification of Breast Cancer Protein Biomarkers at Different Expression Levels in Human Tumors

Liquid chromatography-selected reaction monitoring (LC-SRM) mass spectrometry has developed into a versatile tool for quantification of proteins with a wide range of applications in basic science, translational research, and clinical patient assessment. This strategy uniquely complements traditional pathology approaches, like hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). The multiplexing capabilities offered by mass spectrometry are currently unmatched by other techniques. However, quantification of biomarkers in tissue specimens without the other data obtained from H&E-stained slides or IHC, including tumor cellularity or percentage of positively stained cells inter alia, may not provide as much information that is needed to fully understand tumor biology or properly assess the patient. Therefore, additional characterization of the tissue proteome is needed, which in turn requires the ability to assess protein markers across a wide range of expression levels from a single sample. This protocol provides an example of multiplexed analysis in breast tumor tissue quantifying specific biomarkers, specifically estrogen receptor, progesterone receptor, and the HER2 receptor tyrosine kinase, in combination with other proteins that can report on tissue content and other aspects of tumor biology.