Quantification of Inflammasome Adaptor Protein ASC in Biological Samples by Multiple-Reaction Monitoring Mass Spectrometry

Abstract

Inflammation is an integral component of many diseases, including chronic kidney disease (CKD). ASC (apoptosis-associated speck-like protein containing CARD, also PYCARD) is the key inflammasome adaptor protein in the innate immune response. Since ASC specks, a macromolecular condensate of ASC protein, can be released by inflammasome-activated cells into the extracellular space to amplify inflammatory responses, the ASC protein could be an important biomarker in diagnostic applications. Herein, we describe the development and validation of a multiple reaction monitoring mass spectrometry (MRM-MS) assay for the accurate quantification of ASC in human biospecimens. Limits of detection and quantification for the signature DLLLQALR peptide (used as surrogate for the target ASC protein) were determined by the method of standard addition using synthetic isotope-labeled internal standard (SIS) peptide and urine matrix from a healthy donor (LOQ was 8.25 pM, with a ~ 1000-fold linear range). We further quantified ASC in the urine of CKD patients (8.4 ± 1.3 ng ASC/ml urine, n = 13). ASC was positively correlated with proteinuria and urinary IL-18 in CKD samples but not with urinary creatinine. Unfortunately, the ASC protein is susceptible to degradation, and patient urine that was thawed and refrozen lost 85% of the ASC signal. In summary, the MRM-MS assay provides a robust means to quantify ASC in biological samples, including clinical biospecimens; however, sample collection and storage conditions will have a critical impact on assay reliability.

Keywords: AKI; CKD; MRM-MS; NLRP; PYCARD; SRM-MS; apoptosis-associated speck-like protein containing a CARD; chronic kidney disease; inflammation; urine.