Hi, I am doing mass spectrum to identify the substrates of a ubiquitin-like modification upon stress. I am wondering if I need to do SILAC or just IP-LC-MS/MS if I have antibody against the ubiquitin-like protein.
To my knowledge, IP or Co-IP coupled to LC-MS/MS is good for studying protein-protein interactions.
IP / Co-IP LC-MS/MS can use different quantitative approaches. SILAC is one type. Another common type is LFQ (label-free) approach.
Labeling cells with SILAC (before the IP / Co-IP) allows you to determine the true interacting partners and directly discriminate them from unlabeled environmental contaminants.
Environmental contaminants may lead to an increase in false identifications using LFQ approach. This shortcoming could be overcome by using extra replicates. LFQ is easier to begin with (especially for quick profiling).
Both are acceptable depends on your design.
SILAC is used usually for eukaryotic cells and cell cultures. Else TMT, iTRAQ are also good labels for comparative/quantitative analyses , allowing multiplexing up to at least 8 different sample
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