title: Identification of low-abundance proteins in serum via the isolation of HSP72 complexes.
abstract: Heat shock protein 72 (HSP72) is an intracellular molecular chaperone that is overexpressed in tumor cells, and has also been detected in extracellular regions such as the blood. HSP72 forms complexes with peptides and proteins that are released from tumors. Accordingly, certain HSP72-binding proteins/peptides present in the blood of cancer patients may be derived from tumor cells. In this study, to effectively identify low-abundance proteins/peptides in the blood as tumor markers, we established a method for isolating HSP72-binding proteins/peptides from serum. Nine HSP72-specific monoclonal antibodies were conjugated to N-hydroxysulfosuccinimide-activated Sepharose beads (NHq) and used to isolate HSP72 complexes from serum samples. Precipitated proteins were then identified by LC-MS/MS analysis. Notably, this approach enabled the isolation of low-abundance proteins from serum without albumin removal. Moreover, by subjecting the serum samples of ten patients with multiple myeloma (MM) to NHq analysis, we identified 299 proteins present in MM HSP72 complexes, including 65 intracellular proteins. Among the intracellular proteins detected, 21 were present in all serum samples tested, while 11 were detected in both the conditioned media from cultured multiple myeloma cells and serum from MM patients. These results suggest that the NHq method can be applied to discover candidate tumor markers.
authors.affiliation: Department of Pharmacology, Osaka City University Medical School, Osaka, Japan; Applied Pharmacology and Therapeutics, Osaka City University Medical School, Osaka, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Department of Pharmacology, Osaka City University Medical School, Osaka, Japan. Electronic address: sio@med.osaka-cu.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Shiota
authors.affiliation: Department of Pharmacology, Osaka City University Medical School, Osaka, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Nakao
authors.affiliation: Department of Pharmacology, Osaka City University Medical School, Osaka, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Uemura
authors.affiliation: Department of Pharmacology, Osaka City University Medical School, Osaka, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nishi
authors.affiliation: Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Ohkawa
authors.affiliation: Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Matsumoto
authors.affiliation: Department of Pharmacology, Osaka City University Medical School, Osaka, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: Department of Pharmacology, Osaka City University Medical School, Osaka, Japan.
authors.forename: Mayuko
authors.initials: M
authors.lastname: Osada-Oka
authors.affiliation: Department of Pathology, Osaka City University Medical School, Osaka, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Inagaki
authors.affiliation: Department of Pharmacology, Osaka City University Medical School, Osaka, Japan.
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
authors.forename: Keiichi I
authors.initials: KI
authors.lastname: Nakayama
authors.affiliation: Department of Pathology, Osaka City University Medical School, Osaka, Japan.
authors.forename: Min
authors.initials: M
authors.lastname: Gi
authors.affiliation: Department of Pharmacology, Osaka City University Medical School, Osaka, Japan.
authors.forename: Yasukatsu
authors.initials: Y
authors.lastname: Izumi
authors.affiliation: Applied Pharmacology and Therapeutics, Osaka City University Medical School, Osaka, Japan.
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Miura
authors.affiliation: Department of Pharmacology, Osaka City University Medical School, Osaka, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Iwao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D014408:Biomarkers, Tumor / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D050884:HSP72 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D009101:Multiple Myeloma / Q000097:blood*; D009363:Neoplasm Proteins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D001798:Blood Proteins; D050884:HSP72 Heat-Shock Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins
keywords: Antibody; Heat shock protein 72; Serum; Tumor markers