Liquid TissueTM: proteomic profiling of formalin-fixed tissues

INTRODUCTION Proteomic profiling of diseased and normal tissue by mass spectrometry (MS) is at an early stage, yet it promises to decipher proteomic complexities of the tissue microenvironment and to deliver biomarker information with appropriate pathologic and histologic relevance. Mass spectrometric profiling of the complex cellular proteome obtained from diseased tissue has previously been demonstrated with frozen cancer tissue (1–5). However, the use of frozen tissue for such analysis is problematic for several reasons. These difficulties include collecting sufficient tissue samples for large studies (especially human tissue), the need to design the collection of tissues in a prospective manner, the cumbersome nature of first processing then analyzing frozen tissue, and the effort/expense of storing such tissue on a long-term basis. In contrast, formalin fixation of tissue is a routine process that provides an easily stored archive of tissue that is pathologically well-defined. Extensive archives of animal and human fixed tissue have been collected, assembled, and stored at room temperature for decades; the majority of which contain associated clinical and experimental information representing an extremely valuable untapped reservoir of potential biomarkers. However, current methods of proteomic analysis for formalin-fixed tissue are limited to immunohistochemistry (IHC) (6,7). This methodological limitation is due to the protein cross-linking properties of formalin, which preserves the proteins but renders them insoluble. This process makes the proteins within these samples incompatible with many of the discovery tools used in proteomics today, leaving these fixed archival tissue collections inaccessible for further exploration of biological knowledge (8–12). Expression Pathology has developed a patent pending process, termed Liquid TissueTM, for the extraction of peptides directly from formalin-fixed tissue. This report demonstrates the analysis of peptides obtained from formalin-fixed tissue utilizing the Liquid Tissue process across a variety of MS platforms, including microcapillary reversed-phase liquid chromatography (μRPLC) tandem mass spectrometry (MS/MS), matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF/TOF), and surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF). Results from SELDI-TOF MS analysis reveal tissue-based patterns of protein expression on a variety of chromatographic chip surfaces, while multiple protein identifications were obtained from μRPLC-MS/MS, all from a single Liquid Tissue extract. These results demonstrate that the Liquid Tissue methodology provides the ability to unlock the proteome of the world’s vast reservoir of archived tissue for largescale discovery and validation of biomarkers to improve disease diagnosis and therapy. MATERIALS AND METHODS