title: Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues.
abstract: Formalin-fixed and paraffin-embedded tissues represent the vast majority of archived tissue. Access to such tissue specimens via shotgun-based proteomic analyses may open new avenues for both prospective and retrospective translational research. In this study, we evaluate the effects of fixation time on antigen retrieval for the purposes of shotgun proteomics. For the first time, we demonstrate the capability of a capillary isotachophoresis (CITP)-based proteomic platform for the shotgun proteomic analysis of proteins recovered from FFPE tissues. In comparison to our previous studies utilizing capillary isoelectric focusing, the CITP-based analysis is more robust and increases proteome coverage. In this case, results from three FFPE liver tissues yield a total of 4098 distinct Swiss-Prot identifications at a 1% false-discovery rate. To judge the accuracy of these assignments, immunohistochemistry is performed on a panel of 17 commonly assayed proteins. These proteins span a wide range of protein abundances as inferred from relative quantitation via spectral counting. Among the panel were 4 proteins identified by a single peptide hit, including three clusters of differentiation (CD) markers: CD74, CD117, and CD45. Because single peptide hits are often regarded with skepticism, it is notable that all proteins tested by IHC stained positive.
authors.affiliation: Calibrant Biosystems, 910 Clopper Road, Suite 220N, Gaithersburg, Maryland 20878, USA.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Xu
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Weijie
authors.initials: W
authors.lastname: Wang
authors.forename: Shan-Rong
authors.initials: SR
authors.lastname: Shi
authors.forename: Cheng
authors.initials: C
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Xueping
authors.initials: X
authors.lastname: Fang
authors.forename: Clive R
authors.initials: CR
authors.lastname: Taylor
authors.forename: Cheng S
authors.initials: CS
authors.lastname: Lee
authors.forename: Brian M
authors.initials: BM
authors.lastname: Balgley
mesh_terms: D000941:Antigens / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary; D005557:Formaldehyde; D006801:Humans; D016612:Paraffin Embedding; D040901:Proteomics
chemical_list: D000941:Antigens; D005557:Formaldehyde