Multiplexed Proteome Analysis with Neutron-Encoded Stable Isotope Labeling in Cells and Mice

Overmyer, K.A.; Tyanova, S.; Hebert, A.S.; et al.

To help overcome the challenges of traditional metabolic labeling approaches in qualitative- and quantitative-based proteomic studies, Coon and colleagues developed an alternate labeling approach involving neutron-encoded (NeuCode™) lysine isotopologues. This approach exploits the minor mass differences in the neutron-binding energies of different isotopes of lysine for enhanced peptide identification and protein quantification under high resolution MS. This article details the sample preparation, processing, and analysis protocol required to effectively execute a 3-plex NeuCode experiment for relative protein quantitation of mouse models (SILAM) and cell lines (SILAC) by bottom-up LC-MS/MS (data-dependent acquisition). The application to alternate acquisition strategies and capability for higher multiplexing powers is also discussed.