Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Development of a sensitive and specific targeted mass spectrometry assay for proNGF (#650)

Xiang Li 1 , Sam Faulkner 1 , Matthew D. Dun 1 , Hubert Hondermarck 1
  1. School of Biomedical Sciences and Pharmacy, Hunter Medical Research Institute, The University of Newcastle, Callaghan, NSW 2308, Australia

Background: The precursor for nerve growth factor (proNGF) is emerging as a biomarker and therapeutic target in cancer and neurodegenerative diseases. However, current quantification of proNGF in clinical samples, such as serum and tissues, is based on ELISA and immunohistochemistry and suffers from the limitations of these methods in terms of specificity and sensitivity.   

Aims: To design a sensitive and specific parallel reaction monitoring mass spectrometry (PRM-MS) assay to detect and quantify proNGF in human cell extracts and serum samples.

Methods: PRM-MS was performed using a high-resolution Orbitrap mass spectrometer coupled with nanoflow-liquid chromatography (LC-MS/MS). Initially, tryptic peptide transitions were optimized using stable isotope labelled proNGF peptides in human sera. ProNGF peptides were detected and quantified compared to the abundance of the stable isotope control. Quantification obtained in PRM-MS vs ELISA were also compared.

Results: A proNGF specific peptide at 577.3198++ m/z presenting no similarities with other human proteins was selected. Quantification was based on the corresponding stable isotope control (observed in positive ion mode). Using this assay, proNGF was detected and quantified in cell extracts and human sera. The detection of proNGF was also validated by immunoprecipitation. Although both PRM-MS and ELISA could detect proNGF in the human serum, there was a limited concordance between quantification obtained in PRM-MS vs ELISA.

Conclusion: This study demonstrates that PRM-MS is an efficient technique for the detection and quantification of proNGF in human samples. These results have potential clinical significance in cancer and neurological diseases where proNGF is involved.