title: Tissue proteomics using chemical immobilization and mass spectrometry.
abstract: Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature medium (OCT) or are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomic analyses have been performed on FFPE tissues using advanced mass spectrometry (MS) technologies, high-throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the MS analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Here, we report the development of a method using chemical immobilization of proteins for peptide extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling MS analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all of the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and two-dimensional liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples.
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Punit
authors.initials: P
authors.lastname: Shah
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Bai
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Caitlin
authors.initials: C
authors.lastname: Choi
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Shuang
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Jianying
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Harlan
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Chan
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA. Electronic address: hzhang32@jhmi.edu.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology; D007668:Kidney / Q000378:metabolism; D051379:Mice; D010455:Peptides / Q000032:analysis; D000077489:Piperazine; D010879:Piperazines / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D055767:Immobilized Proteins; D010455:Peptides; D010879:Piperazines; D020543:Proteome; D000077489:Piperazine
keywords: OCT; Polymer contamination; Protein chemical immobilization; Proteomics; Tissues