Liquid Tissue: Proteomic Profiling of Formalin-Fixed Tissues

Identification and quantitation of candidate biomarker proteins in large numbers of individual tissues is required to validate specific proteins, or panels of proteins, for clinical use as diagnostic, prognostic, toxicological, or therapeutic markers. Mass spectrometry (MS) provides an exciting analytical methodology for this purpose. Liquid Tissue MS protein preparation allows researchers to utilize the vast, already existing, collections offormalin-fixed paraffin-embedded (FFPE) tissues for the procurement of peptides and the analysis across a variety of MS platforms

LC/MS-Based Quantitative Proteomic Analysis of Paraffin-Embedded Archival Melanomas Reveals Potential Proteomic Biomarkers Associated with Metastasis

BACKGROUND: Melanoma metastasis status is highly associated with the overall survival of patients; yet, little is known about proteomic changes during melanoma tumor progression. To better understand the changes in protein expression involved in melanoma progression and metastasis, and to identify potential biomarkers, we conducted a global quantitative proteomic analysis on archival metastatic and primary melanomas. METHODOLOGY AND FINDINGS: A total of 16 metastatic and 8 primary cutaneous melanomas were assessed. Proteins were extracted from laser captured microdissected formalin fixed paraffin-embedded archival tissues by liquefying tissue cells. These preparations were analyzed by a LC/MS-based label-free protein quantification method. More than 1500 proteins were identified in the tissue lysates with a peptide ID confidence level of >75%. This approach identified 120 significant changes in protein levels. These proteins were identified from multiple peptides with high confidence identification and were expressed at significantly different levels in metastases as compared with primary melanomas (q-Value<0.05). CONCLUSIONS AND SIGNIFICANCE: The differentially expressed proteins were classified by biological process or mapped into biological system networks, and several proteins were implicated by these analyses as cancer- or metastasis-related. These proteins represent potential biomarkers for tumor progression. The study successfully identified proteins that are differentially expressed in formalin fixed paraffin-embedded specimens of metastatic and primary melanoma

Proteomic Analysis of Formalin-Fixed Prostate Cancer Tissue

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells

Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue

Abstract Background Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies. Methods A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel. Results The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and are consistent with EGFR protein levels in these tumors as previously-reported by western blot and SRM analysis of the matched frozen tissue. In addition, the SRM assay was applied to a collection of histologically-characterized FFPE NSCLC patient tumor tissue where EGFR levels were quantitated from not detected (ND) to 670amol/μg. Conclusions This report describes and evaluates the performance of a robust and reproducible SRM assay designed for measuring EGFR directly in FFPE patient tumor tissue with accuracy at extremely low (attomolar) levels. This assay can be used as part of a complementary or companion diagnostic strategy to support novel therapies currently under development and demonstrates the potential to identify candidates for EGFR-inhibitor therapy, predict treatment outcome, and reveal mechanisms of therapeutic resistance

Location and functional distribution of proteins isolated and identified from melanoma FFPE tissue.

<p>Proteins are categorized by A) cellular origin and, B) biological process, based on the Gene Ontology database.</p

LC/MS-based Label-free Protein Quantification Method.

*<p>Absolute Fold Change.</p>**<p>replicate.</p>***<p>replicate+sample.</p

Metastasis compared to Primary Melanoma.

*<p>Proteins with significant changes greater than 20%.</p

Representative IHC staining of significant proteins found in melanomas.

<p>Anti-PKM1/M2 (rabbit) Ab: A) primary and B) brain metastasis. Anti-HSP90 (rabbit) Ab: C) primary and D) brain metastasis. Anti-MIF (mouse) Ab: E) primary and F) brain metastasis. Anti-STAT1 (mouse) Ab: G) primary and H) brain metastasis. I) Universal negative control rabbit Ig on primary melanoma tissue. J) Universal negative control mouse Ig on brain metastasis tissue. All magnifications were ×100. Scale bars represent 100 µm.</p