Defining TNF-α and IL-1β induced nascent proteins: combining bio-orthogonal non-canonical amino acid tagging and proteomics.
An impediment in the development of new therapeutic strategies for chronic inflammatory diseases is the limited understanding of underlying molecular mechanisms. The objective of this study was to identify newly synthesized (nascent) proteins induced by critical inflammatory cytokines TNF-α and IL-1β in human monocytic THP-1 cells. We optimized methods to combine two different approaches, bio-orthogonal non-canonical amino acid tagging (BONCAT) along with proteomics using isobaric tags (iTRAQ). BONCAT employed the incorporation of l-azidohomoalanine (AHA), an analog of methionine, into TNF-α or IL-1β induced nascent proteins. The AHA-containing nascent proteins were tagged with alkyne-biotin to allow enrichment using avidin affinity purification. The differential expressions of the enriched proteins were further determined using iTRAQ reagents and mass spectrometry (MS). The combination of BONCAT and proteomics represents a unique approach that has uncovered the nascent proteome induced by inflammatory cytokines TNF-α and IL-1β.
Manitoba Centre for Proteomics and Systems Biology, Department of Internal Medicine, Winnipeg, MB, Canada.
Ka-Yee Grace
KY
Choi
Dustin N D
DN
Lippert
Peyman
P
Ezzatti
Neeloffer
N
Mookherjee
D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology; D002460:Cell Line; D006801:Humans; D053583:Interleukin-1beta / Q000276:immunology* / Q000494:pharmacology; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000276:immunology*; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000187:drug effects / Q000276:immunology; D040901:Proteomics; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology* / Q000494:pharmacology
D053583:Interleukin-1beta; D026901:Membrane Transport Proteins; D020543:Proteome; D014409:Tumor Necrosis Factor-alpha; C506098:azidohomoalanine; D000409:Alanine