Multiplexed quantification for data-independent acquisition.

Data-independent acquisition (DIA) strategies provide a sensitive and reproducible alternative to data-dependent acquisition (DDA) methods for large-scale quantitative proteomic analyses. Unfortunately, DIA methods suffer from incompatibility with common multiplexed quantification methods, specifically stable isotope labeling approaches such as isobaric tags and stable isotope labeling of amino acids in cell culture (SILAC). Here we expand the use of neutron-encoded (NeuCode) SILAC to DIA applications (NeuCoDIA), producing a strategy that enables multiplexing within DIA scans without further convoluting the already complex MS(2) spectra. We demonstrate duplex NeuCoDIA analysis of both mixed-ratio (1:1 and 10:1) yeast and mouse embryo myogenesis proteomes. Analysis of the mixed-ratio yeast samples revealed the strong accuracy and precision of our NeuCoDIA method, both of which were comparable to our established MS(1)-based quantification approach. NeuCoDIA also uncovered the dynamic protein changes that occur during myogenic differentiation, demonstrating the feasibility of this methodology for biological applications. We consequently establish DIA quantification of NeuCode SILAC as a useful and practical alternative to DDA-based approaches.