Detection of autoantibodies against cyclophilin A and triosephosphate isomerase in sera from breast cancer patients by proteomic analysis

Abstract

Much interest is presently being shown toward identifying markers for the detection of breast cancer. To detect autoantibodies that could represent diagnostic markers for breast cancer, we comprehensively analyzed serum autoantibodies showing immunoreactivity to proteins in tumor tissues of breast cancer. Tumor tissues were obtained from 40 patients with breast cancer, along with sera from 30 other patients with breast cancer and 22 healthy donors. Proteins from tumor tissues were separated by 2-DE. After blotting onto PVDF membranes, tissue proteins were immunoblotted with sera from patients or healthy donors. By comparing each immunoblot pattern, three immunoreactive spots displayed stronger staining intensity with patient sera than with sera from healthy donors. The matched protein spots on 2-DE gels were digested and used for LC-MS/MS analysis, and identified as cyclophilin A (peptidyl-prolyl cis-trans isomerase A), triosephosphate isomerase and ubiquitin-conjugating enzyme E2N. Immunoblot analysis was then performed using commercially available purified proteins, confirming the specificity of anti-cyclophilin A and anti-triosephosphate isomerase antibodies in sera from patients.

A Multiparametric Serum Marker Panel as a Complementary Test to Mammography for the Diagnosis of Node-Negative Early-Stage Breast Cancer and DCIS in Young Women

“…They are biochemically well-characterized, and many reagents and techniques are available for their detection, simplifying assay development. Until very recently, several specific autoantibodies have been detected in the sera of patients with advanced-stage breast cancer (14,(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58). However, very few studies have attempted to identify and/or evaluate autoantibodies in node-negative earlystage PBC ( 2 cm) or, more importantly, in preinvasive breast cancer, for which mammography's sensitivity is decreased.…”

Mass spectrometry of peptides and proteins from human blood

“…Major protein depletion, solution isoelectric focusing, and 1D PAGE resulted in 575 and 2,890 protein identifications from plasma and serum, respectively . Identifying the targets of immune reactions to proteins from cancer cells, normal self cells, and bacteria has been greatly facilitated by the use of PAGE followed by immunological probing with patient sera and mass spectrometry (Brichory et al, 2001;Hong et al, 2006;Pereira-Faca et al, 2007;Basagana et al, 2008;Bunk et al, 2008;Desmetz et al, 2008;Fujita et al, 2008;Hamrita et al, 2008;Nagashio et al, 2008;Taylor et al, 2008;Zhong et al, 2008;Kim et al, 2008b;Liu et al, 2008b;Forgber et al, 2009;He et al, 2009;Patwa et al, 2009;Rao et al, 2009;Shimada et al, 2009;Tamesa et al, 2009;Yi et al, 2009). A large number of protein isoforms in serum, or EDTA versus citrated plasma, were detected by fluorescent labeling prior to separation of the intact proteins by charge, hydrophobic character and molecular mass (Misek et al, 2005).…”

Immunohistochemical expression of manganese-superoxide dismutase (Mn-SOD) and triose-phosphate isomerase (TPI) in canine mammary gland tumor

“…This dimeric enzyme (molecular weight of~54 kD) is also highly conserved, as sequences are very similar in prokaryotic and eukaryotic organisms (Daar et al 1986). Tamesa et al (2009) showed overexpression of TPI in 47 % of breast cancer patients. In addition, autoantibodies against TPI antigen were detected in both human and canine breast cancers (Tamesa et al 2009;Zamani-Ahmadmahmudi et al 2014).…”