Identification of novel autoantigens via mass spectroscopy-based antibody-mediated identification of autoantigens (MS-AMIDA) using immune thrombocytopenic purpura (ITP) as a model disease

Abstract

Immune thrombocytopenic purpura (ITP) is one of the best characterized autoimmune diseases. Autoantibodies (AABs) against platelet antigens are considered as the diagnostic hallmark of ITP, but are detectable in only 50% of patients. We designed and applied a novel proteomic approach termed Mass Spectroscopy-based Antibody-Mediated Identification of Autoantigens (MS-AMIDA) for platelet antigens. Patients were separated into patients with classical AABs [ITP(+)] and patients without AABs [ITP(−)]. Altogether, 181 potential AAGs were found in ITP(+) and 135 AAGs in ITP(−), with 34 and 23 AAGs reproducibly found in two runs of MS-AMIDA. After subtracting identifiers from the controls, 57 AAGs in ITP(+) and 29 AAGs in ITP(+) remained, with 16 AAGs commonly found in ITP(+) and ITP(−) patients. Label-free quantification (LFQ) revealed 15 potential AAGs that are quantitatively stronger in ITP. Dot blot validation was performed on hexokinase 1 (HK1), E1 pyruvate dehydrogenase (E1-PDH), coagulation factor XIII, filamin A (FLNA), non-muscle myosin 9. Eleven patients were found to have anti-HK1 AABs, one patient had anti-E1-PDH AABs, and two patients had anti-FLNA AABs. Most antigens were of intracellular origin with significant association with actin-cytoskeleton and regulation of programmed cell death. In conclusion, novel AAGs for ITP were identified using MS-AMIDA.