Figure 2.

Mass spectrometry reveals consistent differential phosphorylation changes in NPM harvested from in renal cells, kidney tissue, and urine. Representative tracings used to perform differential phosphoproteomic analysis of purified NPM harvested from the renal cortex of an ischemic human kidney are shown. (A) Liquid chromatography/mass spectrometry base peak peptide profile of an NPM amino acid fragment. (B and C) Liquid chromatography/tandem mass spectrometry (MS/MS) peptide collision-induced dissociation fragment ions providing de novo peptide sequence and specific serine phosphorylation sites represented by loss of −167 D (S-87 + P-80 D). (D) Summary of five differentially NPM-phosphorylated sites altered in a virtually identical manner by ischemic stress in primary murine cells or human proximal tubule epithelial cells (PTECs), murine kidney (mKdy), and human kidney (hKdy) versus control (CTL; murine PTEC, mKdy, and hKdy controls were identical and are shown together). Murine AKI urine (mAKI), human AKI urine (hAKI), and human non-AKI urine (hNonAKI) are also shown. Phosphorylation absent is indicated by a cross; phosphorylation present is indicated by a star. Asterisks show homology between human NPM and murine NPM at residues T²³⁴/T²³² and S²⁴²/S²⁴⁰, respectively. The kinase consensus sequences for each of the five phosphorylation NPM sites are identical between these two species. N/D, not detected.