Quantitative Chemical Proteomics Profiling of de novo Proteins Synthesis during Starvation-Mediated Autophagy

ABSTRACT: Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some non-essential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation, the global protein synthesis is significantly reduced mainly due to suppression of mechanistic target of rapamycin (MTOR), there is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal non-canonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne bearing biotin and AHA’s bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 711 proteins were identified. The characterized functional profiles of these newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the pro-survival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy and cell death. Thus, the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Qingsong Lin  

LAB HEAD: Qingsong Lin

PROVIDER: PXD003546 | Pride | 2016-10-18

REPOSITORIES: Pride