#METABOLOMICS WORKBENCH aferrarini_20200727_085446 DATATRACK_ID:2103 STUDY_ID:ST001434 ANALYSIS_ID:AN002397 PROJECT_ID:PR000985 VERSION 1 CREATED_ON July 28, 2020, 11:10 am #PROJECT PR:PROJECT_TITLE ALDH4A1 is a novel atherosclerosis auto-antigen and a target of protective 6 PR:PROJECT_TITLE antibodies PR:PROJECT_TYPE LC-MS Untargeted Lipidomics PR:PROJECT_SUMMARY Cardiovascular disease (CVD) is the leading cause of mortality in the world, PR:PROJECT_SUMMARY with most CVD deaths resulting from myocardial infarction and stroke. The main PR:PROJECT_SUMMARY cause underlying thrombosis and cardiovascular events is atherosclerosis, an PR:PROJECT_SUMMARY inflammatory disease that can remain asymptomatic for long periods of time. PR:PROJECT_SUMMARY There is an urgent need for new therapeutic and diagnostic options in this area. PR:PROJECT_SUMMARY Atherosclerotic plaques have long been known to contain autoantibodies 1, 2, and PR:PROJECT_SUMMARY there is a well-accepted connection between atherosclerosis and autoimmunity 3. PR:PROJECT_SUMMARY However, the immunogenic trigger and the impact of the autoantibody response PR:PROJECT_SUMMARY during atherosclerosis are not well understood 3, 4, 5. Here we performed a PR:PROJECT_SUMMARY high-throughput single-cell analysis of the atherosclerosis-associated antibody PR:PROJECT_SUMMARY repertoire. Antibody gene sequencing of more than 1700 B cells from atherogenic PR:PROJECT_SUMMARY LDLR-/- mice and control animals identified 56 antibodies expressed by PR:PROJECT_SUMMARY in-vivo-expanded clones of B lymphocytes in the context of atherosclerosis. A PR:PROJECT_SUMMARY third of the expanded antibodies showed reactivity against the atherosclerotic PR:PROJECT_SUMMARY plaque, indicating that various antigens in the lesion can trigger antibody PR:PROJECT_SUMMARY responses. A deep proteomics analysis revealed aldehyde dehydrogenase 4 family PR:PROJECT_SUMMARY member A1 (ALDH4A1), a mitochondrial dehydrogenase involved in proline PR:PROJECT_SUMMARY metabolism, as target antigen of one of these autoantibodies, A12. We show that PR:PROJECT_SUMMARY ALDH4A1 distribution is altered during atherosclerosis and circulating levels of PR:PROJECT_SUMMARY ALDH4A1 are increased in mice and humans with atherosclerosis, supporting the PR:PROJECT_SUMMARY potential use of ALDH4A1 as disease biomarker. A12 antibody infusion into PR:PROJECT_SUMMARY LDLR-/- mice delayed plaque formation and reduced circulating levels of free PR:PROJECT_SUMMARY cholesterol and LDL suggesting that anti-ALDH4A1 antibodies can play a PR:PROJECT_SUMMARY protective role in atherosclerosis progression and might have therapeutic PR:PROJECT_SUMMARY potential. Our study reveals a new auto-antigenic trigger target of the PR:PROJECT_SUMMARY atherosclerosis-associated antibody response and opens new avenues for PR:PROJECT_SUMMARY diagnostic and therapeutic interventions in CVD. PR:INSTITUTE Centro Nacional de Investigaciones Cardiovasculares Carlos III PR:LAST_NAME Ferrarini PR:FIRST_NAME Alessia PR:ADDRESS Calle de Melchor Fernández Almagro, 3, 28029 Madrid PR:EMAIL aferrarini@cnic.es PR:PHONE +34 914 53 12 00 #STUDY ST:STUDY_TITLE Untargeted lipidomics of liver to assess the potential protective role in ST:STUDY_TITLE atherosclerosis progression of A12 antibodies infusion into LDLR-/-mice ST:STUDY_TYPE LC-MS Untargeted Lipidomics ST:STUDY_SUMMARY In order to assess the therapeutic potential of A12 antibodies in ST:STUDY_SUMMARY atherosclerosis, untargeted lipidomics of liver samples was performed. ST:STUDY_SUMMARY LDLR-/-mice were treated with a fully murine version of the A12 antibody ST:STUDY_SUMMARY (mA12-IgG2b), with the isotype control antibody mB1.8-IgG2b (n=16) or with PBS ST:STUDY_SUMMARY as controls. ST:INSTITUTE Centro Nacional de Investigaciones Cardiovasculares Carlos III ST:LAST_NAME Ferrarini ST:FIRST_NAME Alessia ST:ADDRESS Calle de Melchor Fernández Almagro, 3, 28029 Madrid ST:EMAIL aferrarini@cnic.es ST:PHONE +34 914 53 12 00 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 18 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - A12_01 Antibody Group:A12 RAW_FILE_NAME=P_A12_01;N_A12_01 SUBJECT_SAMPLE_FACTORS - A12_02 Antibody Group:A12 RAW_FILE_NAME=P_A12_02;N_A12_02 SUBJECT_SAMPLE_FACTORS - A12_03 Antibody Group:A12 RAW_FILE_NAME=P_A12_03;N_A12_03 SUBJECT_SAMPLE_FACTORS - A12_04 Antibody Group:A12 RAW_FILE_NAME=P_A12_04;N_A12_04 SUBJECT_SAMPLE_FACTORS - A12_05 Antibody Group:A12 RAW_FILE_NAME=P_A12_05;N_A12_05 SUBJECT_SAMPLE_FACTORS - A12_06 Antibody Group:A12 RAW_FILE_NAME=P_A12_06;N_A12_06 SUBJECT_SAMPLE_FACTORS - B18_01 Antibody Group:B1.8 RAW_FILE_NAME=P_B18_01;N_B18_01 SUBJECT_SAMPLE_FACTORS - B18_02 Antibody Group:B1.8 RAW_FILE_NAME=P_B18_02;N_B18_02 SUBJECT_SAMPLE_FACTORS - B18_03 Antibody Group:B1.8 RAW_FILE_NAME=P_B18_03;N_B18_03 SUBJECT_SAMPLE_FACTORS - B18_04 Antibody Group:B1.8 RAW_FILE_NAME=P_B18_04;N_B18_04 SUBJECT_SAMPLE_FACTORS - B18_05 Antibody Group:B1.8 RAW_FILE_NAME=P_B18_05;N_B18_05 SUBJECT_SAMPLE_FACTORS - B18_06 Antibody Group:B1.8 RAW_FILE_NAME=P_B18_06;N_B18_06 SUBJECT_SAMPLE_FACTORS - CTRL_01 Antibody Group:PBS RAW_FILE_NAME=P_CTRL_01;N_CTRL_01 SUBJECT_SAMPLE_FACTORS - CTRL_02 Antibody Group:PBS RAW_FILE_NAME=P_CTRL_02;N_CTRL_02 SUBJECT_SAMPLE_FACTORS - CTRL_03 Antibody Group:PBS RAW_FILE_NAME=P_CTRL_03;N_CTRL_03 SUBJECT_SAMPLE_FACTORS - CTRL_04 Antibody Group:PBS RAW_FILE_NAME=P_CTRL_04;N_CTRL_04 SUBJECT_SAMPLE_FACTORS - CTRL_05 Antibody Group:PBS RAW_FILE_NAME=P_CTRL_05;N_CTRL_05 SUBJECT_SAMPLE_FACTORS - CTRL_06 Antibody Group:PBS RAW_FILE_NAME=P_CTRL_06;N_CTRL_06 SUBJECT_SAMPLE_FACTORS - QC_01 Antibody Group:QC RAW_FILE_NAME=P_QC_01;N_QC_01 SUBJECT_SAMPLE_FACTORS - QC_02 Antibody Group:QC RAW_FILE_NAME=P_QC_02;N_QC_02 SUBJECT_SAMPLE_FACTORS - QC_03 Antibody Group:QC RAW_FILE_NAME=P_QC_03;N_QC_03 SUBJECT_SAMPLE_FACTORS - QC_04 Antibody Group:QC RAW_FILE_NAME=P_QC_04;N_QC_04 SUBJECT_SAMPLE_FACTORS - QC_05 Antibody Group:QC RAW_FILE_NAME=P_QC_05;N_QC_05 SUBJECT_SAMPLE_FACTORS - QC_06 Antibody Group:QC RAW_FILE_NAME=P_QC_06;N_QC_06 SUBJECT_SAMPLE_FACTORS - QC_07 Antibody Group:QC RAW_FILE_NAME=P_QC_07;N_QC_07 #COLLECTION CO:COLLECTION_SUMMARY Liver were collected after mice sacrifice, frozen in liquid N2 and store at CO:COLLECTION_SUMMARY -80°C until the day of analysis. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY LDLR-/-mice under high fat diet were injected with mA12-IgG2b and PBS or a B1-8 TR:TREATMENT_SUMMARY IgG2b isotype as control antibody #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen liver tissues were lysed in 0.5X PBS (sample:solvent 1:10 ratio) SP:SAMPLEPREP_SUMMARY containing 1 mg/mL butylhydroxytoluene (BHT) solution in methanol (proportion SP:SAMPLEPREP_SUMMARY 200 µL per g of tissue), with FastPrep-24 5G instrument (MP Biomedicals, USA). SP:SAMPLEPREP_SUMMARY 25 µL aliquots of homogenate were collected and stored at -80°C until SP:SAMPLEPREP_SUMMARY lipidomic extraction. Tissue homogenates were thawed and extracted with SP:SAMPLEPREP_SUMMARY methyl-tert-butylether (MTBE) as described in [Matyash, V., et al., Lipid SP:SAMPLEPREP_SUMMARY extraction by methyl-tert-butyl ether for high-throughput lipidomics. Journal of SP:SAMPLEPREP_SUMMARY lipid research, 2008. 49(5): p. 1137-46]. 900 µL of organic phase were SP:SAMPLEPREP_SUMMARY dried-out in speedvac and resuspended in 50 µL of ACN:H2O (20:80, v:v) just SP:SAMPLEPREP_SUMMARY before injection. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Agilent mRP-Recovery C18 column (100 × 0.5 mm, 5 µm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MS was operating in full scan mode from 70 to 1700 m/z at 60000 resolution. Data MS:MS_COMMENTS processing was carried-out using Compound Discoverer (ThermoFisher; USA) with MS:MS_COMMENTS the Metaboprofiler node. MS:MS_RESULTS_FILE ST001434_AN002397_Results.txt UNITS:peak area Has m/z:Yes Has RT:No RT units:No RT data #END