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

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 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.

Keywords: LC-MS; MRM; charge derivatization; podocin; podocyturia; preeclampsia.

Figure 1

Sequence of human podocin with marked trypsin cleavage sites determined according to the UniProt databases (entry name PODO_HUMAN). The selected sequences were underlined. Arrow indicates the trypsinolysis site.

Figure 2

Multiple-reaction-monitoring (MRM) analysis of urine sediment tryptic digest from healthy subjects (black line) and pregnant women with diagnosed preeclampsia (red line).

Figure 3

Selected ion monitoring of M(O)IAAEAEK (A) and MIAAEAEK (B) peptide sequences in urine sample tryptic digest from patient with diagnosed preeclampsia (PE). MRM investigation of M(O)IAAEAEK (C,D) and MIAAEAEK (E,F) peptide sequences in urine sediment tryptic digest from pregnant women with diagnosed preeclampsia. MRM transitions corresponding to the oxidized peptide with the M(O)IAAEAEK sequence were not identified.

Figure 4

MRM investigation of ²⁹²MIAAEAEK²⁹⁹ peptide in urine sediment tryptic digest from patient with diagnosed membranous nephropathy (MN) (A,B); focal segmental glomerulosclerosis (FSGS) (C,D); membranoproliferative glomerulonephritis (MPGN) (E,F); and IgA nephropathy (G,H).

Figure 5

MRM analysis of MIAAEAEK(2,4,6-triphenylpyrylium salt (TPP)) peptide conjugate in tryptic digest of urine sediment samples obtained from healthy patient (A,B), patient with PE (C,D), MN (E,F), FSGS (G,H), MPGN (I,J), and IgA nephropathy (IgAN) (K,L).