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

Authors
  • bąchor, remigiusz
  • gąszczyk, dorota
  • panek-laszczyńska, karolina
  • konieczny, andrzej
  • witkiewicz, wojciech
  • stefanowicz, piotr
  • szewczuk, zbigniew
Publication Date
May 02, 2020
Identifiers
DOI: 10.3390/ijms21093225
OAI: oai:mdpi.com:/1422-0067/21/9/3225/
Source
MDPI
Keywords
Language
English
License
Green
External links

Abstract

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 &epsilon / 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.

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