Using instrument logic to improve identifications in low stoichiometry SILAC/TMT experiments

Pulsed SILAC approaches allow measurement of protein dynamics, including protein translation and degradation. However, its use in quantifying acute changes has been limited due the low labeled peptide stoichiometry. Here, we describe the use of instrument logic to select peaks of interest via targeted mass differences (TMD) for overcoming this limitation. Comparing peptides artificially mixed at low heavy-to-light stoichiometry measured using standard data dependent acquisition with or without TMD revealed 2-3 fold increases in identification without significant loss in quantification precision for both MS2 and MS3 methods. Our benchmarked method approach increases throughput by reducing the necessary machine time. We anticipate that all pulsed SILAC measurements, if combined with TMT or not, would greatly benefit from instrument logic based approaches.