Detection of Podocin in Human Urine Sediment Samples by Charge Derivatization and LC-MS-MRM Method

• Main Authors: Remigiusz Bąchor, Dorota Gąszczyk, Karolina Panek-Laszczyńska, Andrzej Konieczny, Wojciech Witkiewicz, Piotr Stefanowicz, Zbigniew Szewczuk
• Format: Article
• Language: English
• Published: MDPI AG 2020-05-01
• Series: International Journal of Molecular Sciences
• Subjects: podocin; podocyturia; preeclampsia; charge derivatization; LC-MS; MRM
• Online Access: https://www.mdpi.com/1422-0067/21/9/3225

Detection of podocytes in urine might serve as a useful diagnostic tool in both primary and secondary glomerular diseases. The utility of podocyturia has been confirmed for both pre-eclampsia and glomerulonephritis. Here, we present a new and sensitive method for qualitative LC-MS-multiple-reaction-monitoring (MRM) analysis of podocin, serving as a podocyturia biomarker in urine sediments. The following podocin tryptic peptides with the ¹⁶⁹LQTLEIPFHEIVTK¹⁸², ²¹³AVQFLVQTTMK²²³, ²⁴⁰SIAQDAK²⁴⁶, and ²⁹²MIAAEAEK²⁹⁹ sequences were applied as a model. The selective chemical derivatization of the ε amino group of C-terminal lysine residue in tryptic peptides, by 2,4,6-triphenylpyrylium salt (TPP) as a fixed charge tag, was employed to increase the ionization efficiency, in routine ESI-MS analysis. Additionally, the generation of a reporter ion, in the form of a protonated 2,4,6-triphenylpyridinium cation, makes the derivatized peptide analysis in the MRM mode unambiguous. Identification of derivatized and non-derivatized peptides were performed, and the obtained results suggest that the peptide with the ²⁹²MIAAEAEK²⁹⁹ sequence may serve as a marker of podocyturia.