Dataset Information

HCV Cryoprecipitate LC-MS/MS with iTRAQ Quantification

ABSTRACT: The discovery of hepatitis C virus (HCV) in 1989 revealed the virus as the etiology of 40%-90% of the “essential” mixed cryoglobulinemia, where immune complex forms deposit called cryoprecipitate at temperatures below 37 °C. Cryoprecipitate constitutes monoclonal IgM and polyclonal IgG, some of which has reactivity against HCV core and NS3 epitopes. Resultant immune complex is considered entrapped on microvascular endothelium via C1q receptor, leading to complement activation and organ injury presenting predominantly as dermopathy, peripheral neuropathy and nephropathy. However, currently little is known on whether auto-reactive, cold-precipitating IgG components enriched in cryoprecipitate may play some role on the deposition of immune complex and subsequent complement-mediated injury of specific organs. Recently, with the advent of high-throughput immune repertoire sequencing and mass spectrometry, technical feasibility is growing to delineate antibodies of interest and their sequences directly from serum. To date, vast majority of studies actually utilized the antigen column for affinity purification of antibodies of interest, although this strategy is not applicable to disease entity with unknown antigen involvement. In such cases, disease-specific and organ-specific immune deposits may be a good alternative source of etiological antibodies. Herein, targeting HCV cryoglobulinemic vasculitis as a model, we conducted a proof-of-concept study aiming at characterizing the IgG components most prone to cryoprecipitation. To this end, we longitudinally studied one patient with cryoglobulinemic vasculitis with chronic HCV infection. After obtaining informed consent, cryoprecipitate and supernatant were separated from peripheral blood sample. Fab fragments from Protein G-purified IgG were recovered after papain digestion for isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics. Simultaneously, total RNA was isolated from peripheral blood, and immunoglobulin heavy chain variable region (IGHV) was PCR-amplified with unique molecular identifier (UMI) strategy to construct a personal IGHV sequence library of immunoglobulin variable region. Sequencing output from MiSeq was bioinformatically converted into mass spectrometry database. Search was performed using MaxQuant software.

OTHER RELATED OMICS DATASETS IN: PRJDB4353MSV000085207

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

DISEASE(S): Cryoglobulinemia,Hepatitis C

SUBMITTER: Masato Ogishi

LAB HEAD: Hiroshi Yotsuyanagi

PROVIDER: PXD002475 | Pride | 2016-07-14

REPOSITORIES: Pride

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Publications

Delineation of autoantibody repertoire through differential proteogenomics in hepatitis C virus-induced cryoglobulinemia.

Ogishi Masato M Yotsuyanagi Hiroshi H Moriya Kyoji K Koike Kazuhiko K

Scientific reports 20160712

Antibodies cross-reactive to pathogens and autoantigens are considered pivotal in both infection control and accompanying autoimmunity. However, the pathogenic roles of autoantibodies largely remain elusive without a priori knowledge of disease-specific autoantigens. Here, through a novel quantitative proteogenomics approach, we demonstrated a successful identification of immunoglobulin variable heavy chain (VH) sequences highly enriched in pathological immune complex from clinical specimens obt ...[more]

PMID: 27403724

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Project description:The discovery of hepatitis C virus (HCV) in 1989 revealed the virus as the etiology of 40%-90% of the “essential” mixed cryoglobulinemia, where immune complex forms deposit called cryoprecipitate at temperatures below 37 °C. Cryoprecipitate constitutes monoclonal IgM and polyclonal IgG, some of which has reactivity against HCV core and NS3 epitopes. Resultant immune complex is considered entrapped on microvascular endothelium via C1q receptor, leading to complement activation and organ injury presenting predominantly as dermopathy, peripheral neuropathy and nephropathy. However, currently little is known on whether auto-reactive, cold-precipitating IgG components enriched in cryoprecipitate may play some role on the deposition of immune complex and subsequent complement-mediated injury of specific organs. Recently, with the advent of high-throughput immune repertoire sequencing and mass spectrometry, technical feasibility is growing to delineate antibodies of interest and their sequences directly from serum. To date, vast majority of studies actually utilized the antigen column for affinity purification of antibodies of interest, although this strategy is not applicable to disease entity with unknown antigen involvement. In such cases, disease-specific and organ-specific immune deposits may be a good alternative source of etiological antibodies. Herein, targeting HCV cryoglobulinemic vasculitis as a model, we conducted a proof-of-concept study aiming at characterizing the IgG components most prone to cryoprecipitation. To this end, we longitudinally studied one patient with cryoglobulinemic vasculitis with chronic HCV infection. After obtaining informed consent, cryoprecipitate and supernatant were separated from peripheral blood sample. Fab fragments from Protein G-purified IgG were recovered after papain digestion for isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics. Simultaneously, total RNA was isolated from peripheral blood, and immunoglobulin heavy chain variable region (IGHV) was PCR-amplified with unique molecular identifier (UMI) strategy to construct a personal IGHV sequence library of immunoglobulin variable region. Sequencing output from MiSeq was bioinformatically converted into mass spectrometry database. Search was performed using MaxQuant software.

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