Abstract
The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.
Highlights
Peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues
We developed and evaluated an selected reaction monitoring (SRM) assay for the quantification of six peptides of the human receptor tyrosine-protein kinase erbB-2 (HER2) and compared the obtained results with those of standard methods used in clinical pathology, namely IHC and fluorescence in situ hybridization (FISH)
Development of the Analytical Method—In order to identify the HER2 peptides generated from biological material containing the protein of interest, untargeted LC-MS analysis was performed on peptide extracts from untreated and formalinfixed cell lines (SKBR-3 and BT-474)
Summary
The untargeted mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [24] via the PRIDE partner repository with the data set identifier PXD002281 and under the project name “Identification of HER2 peptides for quantification in formalin-fixed paraffin-embedded breast cancer tissues”. Ordinal multinomial logistic regression: For each sample i, the IHC score (or FISH category) Yi and the normalized HER2 peptide concentration xi is determined. The relationship between the IHC score and the normalized HER2 peptide concentration is adequately modeled by an ordinal multinomial logistic regression, which was performed as previously described [35, 36]. This model aims to predict the probability of association of sample i to be assigned to category j based on xi, or pij ϭ P(Yi ϭ j). The probability P(Y ϭ j) is estimated from P(Y ϭ j) ϭ P(Y £ j) Ϫ P(Y £ j Ϫ 1) [37]
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