Neutrophil gelatinase associated lipocalin (NGAL) is a protein that was found to be overexpressed in acute kidney injury (AKI). The rise in NGAL concentration, both in urine or plasma, appears earlier than for other classical renal function markers such as serum creatinine, thus making it a suitable marker for early pathology detection. The aim of this study was to develop a method involving tryptic digestion, solid phase extraction and LC-MS/MS analysis to analyze NGAL in plasma medium using an isotope labeled surrogate protein, containing NGAL signature tags, as internal standard (QPrEST). The method was validated for the analysis of NGAL in an analytical range from 50 to 1250 ng/mL using two different proteotypic peptides. The method was further used to quantify the NGAL in human plasma samples for whom elevated NGAL values were expected. NGAL values were between 190.8 and 242.6 ng/mL for control group and between 228.1 and 3526.2 ng/mL for patient group. This study proved that the selection of the right internal standard is of utmost importance in targeted proteomics studies as the digestion steps might cause high variability. This study also confirmed that, although NGAL is highly resistant to proteases such as trypsin, the method could be fully validated according to FDA guidelines and subsequently used to assess NGAL levels in patient plasma with high analytical confidence.
Keywords: AKI; NGAL; Targeted proteomics; UHPLC-MS/MS.
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