Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS

Abstract

There exists a need for robust approaches for tandem mass spectrometry (MS/MS)-based identification of proteins in formalin-fixed paraffin-embedded (FFPE) material. We demonstrate herein the identification of proteins in FFPE material using enzymatic cleavage for extraction of peptides from the FFPE specimen and liquid chromatography (LC) followed by MS/MS. We identified 324 proteins from a 3-year-old FFPE cell-block of a human lymphoma cell line. The identified proteins were assigned to the membrane, cytosol and nucleus, with diverse cellular functions. The results were comparable to those obtained with lysates from a fresh specimen of the lymphoma cell line. Western blotting analysis and immunofluorescence microscopy confirmed the expression of selected proteins. The functional significance of one protein (PKC g) was validated using a PKC inhibitory peptide which resulted in lymphoma cell death in vitro. The ability to identify proteins from FFPE specimens has significant implications for MS/MS-based proteomics of vast repositories of archival primary tissue samples for disease-related discovery research. Laboratory Investigation ( Keywords: bottom-up proteomics; formalin fixed; paraffin embedded; tandem mass spectrometry Formalin fixation and tissue embedding in paraffin wax is a universal approach for tissue processing prior to light microscopic evaluation. A major advantage afforded by formalin-fixed paraffin-embedded (FFPE) specimens is the preservation of cellular and architectural morphologic detail in tissue sections. 1 The standard buffered formalin fixative in which biopsy specimens are processed is an aqueous solution containing 37% formaldehyde and 10-15% methyl alcohol. Formaldehyde is a highly reactive dipolar compound that results in the formation of protein-nucleic acid and proteinprotein crosslinks in vitro. [1][2][3][4][5] FFPE specimens have not been routinely used for global mass spectrometry-based proteomic studies. This relates to the fact that formaldehyde-induced crosslinking also renders proteins relatively insoluble and unsuitable for routine biochemical extraction and analysis. This crosslinking effect has precluded protein extraction from FFPE for routine Western blotting analysis of protein expression. 2 Thus, while recently developed enzymatic and heatinduced antigen retrieval methods have facilitated the immunohistologic detection of proteins in tissue, many proteins are still not detectable using these approaches. 6,7 In addition, proteins lacking widely available antibodies to their formalin-resistant epitopes are largely undetectable by immunohistochemistry. Although recent advances in molecular biotechnology have permitted the global analysis of proteins expressed in various cellular and tissue systems, the diminished capacity to extract intact proteins has largely precluded the proteomic analysis of FFPE samples with definitive identification of the proteins present within cells and tissues in biopsy material.The development of strategies to permit utilization of the universa...

Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry

“…5). We attribute the poor outcome of Glu-C digests for identifying phosphopeptides to the fact that although the average length of peptides generated is indeed in a favorable range (between trypsin and Lys-C), the fragmentation in ETD is impaired, as noted previously for CID (15). database.…”

‘Tissue surrogates' as a model for archival formalin-fixed paraffin-embedded tissues

“…Although these results are encouraging, there are a number of challenges that must be addressed to develop better and more reproducible techniques for extracting and identifying proteins useful for proteomic analysis from archival FFPE tissue. The study by Crockett et al 5 perhaps best illustrates the current state of our ability to use archival FFPE tissues for proteomic studies. Liquid chromatographytandem mass spectrometry was used to compare proteins identified in a fresh cell lysate to the same cells processed as an FFPE cell plug.…”

Protein unlocking procedures of formalin‐fixed paraffin‐embedded tissues: Application to MALDI‐TOF Imaging MS investigations

“…[3] demonstrated that the proteins can be extracted from ethanol-fixed and paraffin-embedded tissue, and resolved by 2-DE separation. Recently Shi et al [4] obtained a satisfying overlapping between proteins identified from FFPE and frozen tissues and encouraging results are reported also by Prieto et al [5] and Crockett et al [6] that identified proteins from FFPE enzymatically digested tissues. However, few studies have been pursuing MALDI-TOF imaging MS (MALDI-IMS), the most challenging proteomics analysis application on FFPE section.…”