Optimized Protocol for Quantitative Multiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues

Abstract

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R(2) = 0.94) and immuno-MRM (R(2) = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.

Keywords: FFPE; archived tissue; immunoaffinity enrichment; mass spectrometry; multiple reaction monitoring; peptide assays; targeted proteomics.

Figures

Figure 1. Test battery approach for finding the optimal process for protein extraction and digestion from FFPE

A. A test battery approach for evaluation of FFPE processing protocols using an ER+/HER2+ breast cancer specimen cut in 10 μm sections. Each combination of protein extraction and protein digestion protocols was performed in triplicate (using a single process replicate from 3 separate days, yielding 27 total samples). Performance of the protocols was evaluated by LC-MS/MS ‘shotgun’ analysis and LC-MRM targeted analysis. B. Performance of the method was compared in analysis of FFPE and frozen tissue prepared from a single specimen. Alternating sections were used for fixation or freshly frozen. Direct LC-MRM and immuno-MRM assays were applied for quantitative analysis of targeted peptides.

Figure 2. Evaluation of processes for quantitative proteomics on FFPE

Results are from the analysis of an FFPE, ER+/HER2+ breast cancer. Each sample consists of three 10 μm sections. All error bars reflect the standard deviation of three analytical process replicates (i.e. de-paraffinization, protein extraction, digestion, enrichment, analysis performed on three separate days). A. Protein yield measured by micro-BCA assay. B. Unique peptide identifications obtained from shotgun LC-MS/MS results. Equal amount of protein (2 μg) was loaded on-column for each process. C. Trypsin digestion efficiency and fidelity measured by the percentage of identifications containing missed cleavages (internal K or R) or mis-cleavages (non-tryptic cut site). D. LC-MRM results showing the number of observable peptides (>LOD in 2 out of 3 replicates). Heavy peptide standards were added to1 μg of the digested lysates at 10 fmol/μg protein and analyzed in process triplicate. E. LC-MRM results showing the distribution of CVs for observable peptides. F. Distribution of peptide amounts measured by LC-MRM. Box plots show the median (bar), inner quartiles (box), 5-95th percentiles (line), and outliers (points).

Figure 3. Characterization of assays in frozen and FFPE matrices

LC-MRM and immuno-MRM assays were characterized by reverse response curves in frozen or FFPE tissue extracts. A. Example response curves for the peptide GLQSLPTHDPSPLQR (ERBB2) measured by LC-MRM. Responses measured in FFPE and frozen tissues are overlaid, and curves are plotted on a log10 scale. Error bars are the standard deviation of complete process triplicate measurements. B. Example response curves for the peptide GLQSLPTHDPSPLQR (ERBB2) measured by immuno-MRM. Responses measured in FFPE and frozen tissues are overlaid, and curves are plotted on a log10 scale. Error bars are the standard deviation of complete process triplicate measurements. C. Distribution of LLOQs and CVs measured in FFPE and frozen tissue for all analytes detected by LC-MRM. D. Distribution of LLOQs and CVs measured in FFPE and frozen tissues for all analytes detected by immuno-MRM. Box plots show the median (bar), inner quartiles (box), 5-95th percentiles (line), and outliers (points).

Figure 4. Comparison of protein quantification in matched frozen and FFPE biospecimens

Three individual breast cancer tissues were sectioned and analyzed by multiplexed LC-MRM (targeting 512 peptide analytes) and immuno-MRM (targeting 42 peptide analytes) assays. Each sample was analyzed in process triplicate, and each replicate consisted of extraction, digestion, and enrichment performed on three separate days. A. Protein recovery from matched FFPE and frozen samples measured by BCA assay. B. Overlap in number of peptides detected in quantitative LC-MRM and immuno-MRM assays in matched FFPE and frozen tissue specimens. C. Scatter plot of endogenous peptide concentrations (n= 356) determined by MRM measurements made in the matched FFPE and frozen tissue. D. Repeatability (CV) of immuno-MRM and LC-MRM assays applied to FFPE and frozen tissue, each box plot represents three samples measured on three days (n = 9). E. Scatter plot of endogenous peptides (n= 49) with overlapping immuno-MRM and LC-MRM measurements in FFPE (yellow) and frozen (blue) tissues. Error bars are the standard deviation of triplicate measurements.