MSACL 2019 EU Abstract

Multiplexing Kidney Injury and Kidney Specific Biomarkers in Urine Using MS-based Bottom-Up Proteomics: A Robust and Feasible Approach

T.T. van DUIJL, L.R. RUHAAK, N.P.M. SMIT, C.M. COBBAERT
Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands

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Tirsa van Duijl (Presenter) Leiden University Medical Center (LUMC)

Presenter Bio: After obtaining my master’s degree in Bio-Pharmaceutical Sciences at Leiden University, I was pleased to join the research group of prof. C.M. Cobbaert at the Clinical Chemistry department of the Leiden University Medical Center (LUMC) as a PhD candidate. My ongoing research is focused on the search for a biomarker panel for kidney damage and the development of an LC-MS based assay for biomarker quantification in the clinical laboratory.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

INTRODUCTION:Acute Kidney Injury (AKI) is a frequent complication in hospitalized patients. Early detection of AKI is an unmet clinical need because current markers are delayed markers of functional kidney loss. Early kidney damage biomarkers such as neutrophil gelatinase-associated lipocalin (NGAL), tissue inhibitor of metalloproteinases 2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) in urine are under investigation for early AKI detection and are typically measured by immunoassays. To increase specificity for kidney injury and to obtain information on the underlying pathology, kidney-specific proteins (e.g. uromodulin) and AKI biomarkers may be measured in a multiplex method. Here we aim to develop a multiplex LC-MRM-MS based method that is suitable for AKI biomarker quantitation.
METHODS:Urines from healthy individuals and from kidney transplant patients were collected, deidentified, pooled and stored at -80˚C till analysis. Samples were heated to inactivate urinary proteases and stable-isotope labelled peptides were added as IS. After denaturation, reduction and alkylation, proteins were tryptically digested and samples were desalted by HLB SPE. For immunoprecipitation (IP), antibodies on beads were incubated with urine. After elution, isolated proteins were digested. An Agilent 1290 infinity LC system coupled to an Agilent 6495 QQQ-MS was used to develop an LC-MRM-MS method for NGAL, TIMP-2, IGFBP7 and uromodulin. At least 3 candidate peptides were identified per protein by at least 3 fragments using product-ion scans and MRM transitions were developed.
RESULTS:Tryptic peptides from NGAL (ITLDEYWR, MYATIYELK, WYVVGLAGNAILR), TIMP-2 (EYLIAGK, FFACcamIK, IQYEIK), IGFBP7 (TELLPGDR, ITVVDALHEIPVK, DNLAIQTR) and uromodulin (YFIIQDR, VGGTMFTVR, VLNLGPITR) were identified. In one LC-MRM-MS run, all peptides from urinary NGAL, TIMP-2, IGFBP7 and uromodulin elute within 8 min. For LC-MS instrument monitoring, a system suitability test containing synthetic peptides and their labelled counterparts was developed with within- and between-run CVs <5% and carryover <1%. Direct protein digestion and LC-MRM-MS measurement from urine was hampered by urinary matrix effects. IP was needed for robust quantitation of TIMP-2 and IGFBP7 and the mean IP recovery was 85±1% and 97±4%, respectively. IP resulted in good measurement linearity (r2 = 0.99 at 0.5 – 15.5 fmol), precision (CV <5%) and S/N (S/N >100) for all peptides.
CONCLUSION:AKI biomarkers and kidney-specific uromodulin were included in a multiplex LC-MRM-MS method using bottom-up proteomics. Here we show that IP in combination with LC-MRM-MS allows accurate quantitation of low abundant proteins in the pmol/L range and overcomes urinary matrix effects. This strategy demonstrates the feasibility of developing an extended biomarker panel, in which the biomarker selection is guided by nephrologists’ unmet clinical needs, to enable early detection of AKI in hospitalized patients.