Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS.
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 eta) 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.
Crockett
David K
DK
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84132, USA.
Lin
Zhaosheng
Z
Vaughn
Cecily P
CP
Lim
Megan S
MS
Elenitoba-Johnson
Kojo S J
KS
D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D005404:Fixatives / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D016393:Lymphoma, B-Cell / Q000473:pathology; D013058:Mass Spectrometry; D009363:Neoplasm Proteins / Q000032:analysis*; D016612:Paraffin Embedding
D005404:Fixatives; D009363:Neoplasm Proteins; D005557:Formaldehyde