HILAQ: A Novel Strategy for Newly Synthesized Protein Quantification

Here
we describe a new strategy, HILAQ (Heavy Isotope Labeled Azidohomoalanine
Quantification), to rapidly quantify the molecular vulnerability profile
to oxytosis, which is an oxidative stress-induced programed cell death
pathway that has been reported to be involved in aging and neurodegenerative
diseases. HILAQ was able to quantify 1962 newly synthesized proteins
(NSPs) after 1 h of pulse labeling in HEK293T cell line, while 353
proteins were quantified using the previously published QuaNCAT protocol.
HILAQ was successfully applied to the HT22 oxytosis model. 226 proteins
were found to have a two-fold change in abundance, and 108 proteins
were enriched in the cell death pathway, demonstrating the utility
of HT22 cells as a tool to study the molecular details of cell death
involved in neurodegenerative diseases. The HILAQ strategy simplifies
the analysis of newly synthesized proteomes through the use of isobaric
labels and achieves higher sensitivity than previously published methods.