1. Application of Selected Reaction Monitoring for Multiplex Quantification of Clinically Validated Biomarkers in Formalin-Fixed, Paraffin-Embedded Tumor Tissue 
Todd Hembrough, Sheeno Thyparambil, Wei-Li Liao, Marlene M. Darfler, Joseph Abdo, Kathleen M. Bengali, Stephen M. Hewitt, Richard A. Bender, David B. Krizman, Jon Burrows 
The Journal of Molecular Diagnostics 
Volume 15, Issue 4, Pages (July 2013)
DOI: /j.jmoldx
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Liquid Tissue-SRM work flow for analysis of proteins from FFPE tissue. Tissues are divided into sections onto Director microdissection slides, deparaffinized, and counterstained with hematoxylin. Areas of interest are identified by a pathologist, and laser microdissection is performed to collect the specified cellular material. The Liquid Tissue protocol is performed on the microdissected tissue to achieve a completely soluble peptide lysate. SRM analysis is then conducted using labeled, synthetic, internal standard peptides, allowing for extremely accurate measurement of native, endogenous peptides.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Development of four SRM assays: IGF-1R-SRM (A), EGFR-SRM (B), HER2-SRM (C), and HER3-SRM (D). These assays showed the standard curve in Pfu matrix (top row), standard curve in eukaryotic cell matrix (second row), total ion chromatograms (third row), and transition ion set (bottom row) for each assay.
The standard curve for EGFR-SRM has been published previously and is reproduced herein with permission from the BioMed Central.29
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Characterization of three SRM assays: IGF-1R-SRM (A), EGFR-SRM (B), and HER2-SRM (C). A-C: Correlation with immunoassay with all results standardized per μg of total cellular protein. ECL (A and C) and ELISA (B) using FFPE cultured cell lines.
The ELISA correlation curve and table for EGFR-SRM have been published previously and are reproduced herein with permission from the BioMed Central.29
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
HER2-SRM analysis in breast cancer patient tumors. Quantitative HER2-SRM analysis of 10 FFPE breast cancer patient tumors across a 0 to 3+ range of IHC scores (B). Results indicate HER2 protein expression measured by SRM (A) across approximately two orders of magnitude. All results are presented as means, with error bars representing SDs for triplicate measurements.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Figure 5
HER2-SRM analysis in breast cancer patient tumors. Quantitative HER2-SRM analysis of 19 FFPE breast cancer patient tumors (B). All tumors were stained for HER2, and all show an IHC of 3+ (A). SRM analysis indicates HER2 protein is expressed across greater than two orders of magnitude within the IHC of 3+ sample set. All results are presented as means, with error bars representing SDs for triplicate measurements.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7. Figure 6
Multiplexed measurement of HER2, HER3, EGFR, and IGF-1R in FFPE tumor samples obtained from 19 patients with advanced breast carcinoma who had been treated with the anti-HER2 antibody, trastuzumab. All results are presented as means, with error bars representing SDs for triplicate measurements. A: Results presented for HER2 and HER3 only. B: Results presented for HER2 and EGFR only. C: Results presented for HER2 and IGF-1R only. D: Results are presented for all four biomarkers.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions