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

Pulsed Azidohomoalanine Labeling in Mammals (PALM) Detects Changes in Liver-Specific LKB1 Knockout Mice

Quantification of proteomes by mass spectrometry has proven to be useful to study human pathology recapitulated in cellular or animal models of disease. Enriching and quantifying newly synthesized proteins (NSPs) at set time points by mass spectrometry has the potential to identify important early regulatory or expression changes associated with disease states or perturbations. NSP can be enriched from proteomes by employing pulsed introduction of the noncanonical amino acid, azidohomoalanine (AHA). We demonstrate that pulsed introduction of AHA in the feed of mice can label and identify NSP from multiple tissues. Furthermore, we quantitate differences in new protein expression resulting from CRE-LOX initiated knockout of LKB1 in mouse livers. Overall, the PALM strategy allows for the first time in vivo labeling of mouse tissues to differentiate protein synthesis rates at discrete time points