HER2 protein level was quantified by SRM mass spectrometry as previously described [9–11 (link)]. Briefly, one section was cut for hematoxylin and eosin (H&E) staining, and multiple sections were cut on to Director® microdissection slides and stained with eosin. The H&E was used to guide tumor area selection on the slide sections, which were then microdissected (Molecular Machines & Industries, Eching, Germany). Collected tumor tissue was solubilized using Liquid Tissue® according to manufacturer’s instructions. Total protein concentration from each sample was measured by a micro bicinchoninic acid assay (Thermo Fisher Scientific Inc, Waltham, MA).
A mixture of stable isotope-labeled synthetic peptides was added to the liquefied tumor samples as internal standards. All samples were analyzed in triplicate using a triple quadrupole mass spectrometer (TSQ Quantiva™, Thermo Scientific, Waltham, MA) interfacing with a nanoACQUITY liquid chromatography system (Waters Corporation, Milford, MA). A 100 µm inner diameter chromatographic column packed with C18 resin (ProntoSIL 200-5-C18AQ; Bischoff Chromatography, Germany) was used for peptide separation before mass spectrometry analysis. For protein quantitation, peak areas from each endogenous and internal standard peptide were calculated and ratios were determined using PinPoint 1.3 (Thermo Scientific, Waltham, MA).
A mixture of stable isotope-labeled synthetic peptides was added to the liquefied tumor samples as internal standards. All samples were analyzed in triplicate using a triple quadrupole mass spectrometer (TSQ Quantiva™, Thermo Scientific, Waltham, MA) interfacing with a nanoACQUITY liquid chromatography system (Waters Corporation, Milford, MA). A 100 µm inner diameter chromatographic column packed with C18 resin (ProntoSIL 200-5-C18AQ; Bischoff Chromatography, Germany) was used for peptide separation before mass spectrometry analysis. For protein quantitation, peak areas from each endogenous and internal standard peptide were calculated and ratios were determined using PinPoint 1.3 (Thermo Scientific, Waltham, MA).