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

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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.

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Abbreviations

AKI:

acute kidney injury

AMBIC:

ammonium bicarbonate

ASC:

apoptosis-associated speck-like protein containing a CARD

CKD:

chronic kidney disease

CoV:

coefficient of variance

Cr:

creatinine;

DTT:

dithiothreitol

IL:

interleukin

LOD:

lower limit of detection

LOQ:

lower limit of quantification

MS:

mass spectrometry

MRM:

multiple reaction monitoring

SIS:

stable isotope-labeled internal standard

SRM:

selected reaction monitoring

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Acknowledgments

The authors acknowledge Jaye Platnich and Jae Hyun Chung for their technical support. Additional technical support was provided by Shu Li and the Biobank for the Molecular Classification of Kidney Disease at the Snyder Institute for Chronic Diseases. J.A.M. was the holder of a Marie (Sklodowska-) Curie International Incoming Fellowship from the European Commission. D.A.M. holds a Tier II Canada Research Chair. A.L. held a Postdoctoral Scholarship Award from Alberta Innovates—Health Solutions.

Funding

Research was supported by the Canadian Institutes of Health Research (CIHR) Health Challenges in Chronic Disease Signature Initiative (#THC-13523), the Canadian National Transplantation Research Program (CNTRP), and the Canada Foundation for Innovation.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3280 Hospital Drive N.W, Calgary, AB, T2N 4Z6, Canada

    Annegret Ulke-Lemée & Justin A. MacDonald

  2. Department of Medicine, Cumming School of Medicine, University of Calgary, 3280 Hospital Drive NW, Calgary, T2N 4Z6, Alberta, Canada

    Arthur Lau, Michelle C. Nelson, Matthew T. James & Daniel A. Muruve

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  1. Annegret Ulke-Lemée
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Contributions

A.U.-L. designed and performed the MRM-MS experiments and analyzed the data. A.L. generated HEK-293T and THP-1 samples for analysis and measured IL-18 levels in patient urine by ELISA. J.A.M. secured funding and conceived the project. M.C.N. acted as the clinical research coordinator, collected urinary specimens, and managed the Biobank for the Molecular Classification of Kidney Disease. D.A.M. and M.T.J. provided expertise and led the clinical CKD study. J.A.M. wrote the manuscript and manufactured the figures with assistance from A.U.-L. All authors have given approval to the final version of the manuscript.

Corresponding author

Correspondence to Justin A. MacDonald.

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Competing Financial Interests

J.A.M. and D.A.M. are co-founders and hold equity positions in Arch Biopartners Inc.

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Ulke-Lemée, A., Lau, A., Nelson, M.C. et al. Quantification of Inflammasome Adaptor Protein ASC in Biological Samples by Multiple-Reaction Monitoring Mass Spectrometry. Inflammation 41, 1396–1408 (2018). https://doi.org/10.1007/s10753-018-0787-6

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