Measuring autophagic flux by quantitative mass spectrometry. Bertina Telusma1, Jean-Claude Farre2, Suresh Subramani2, Joey Davis1 1Department of Biology, Massachusetts Institute of Technology; 2Molecular Biology, Division of Biological Sciences, University of California, San Diego. Autophagy is an essential, well-conserved catabolic process known to deliver a variety of proteins and organelles to the lysosome/vacuole for degradation. Despite extensive study, a comprehensive profile of autophagic substrates has not been reported and the relative contributions of non-specific (bulk) autophagy vs. selective autophagy to such degradation is underexplored. Here, we develop and employ a novel autophagic assay that utilizes stable isotope pulse-labeling and mass spectrometry to quantify bulk or selective autophagic degradation on a protein-by-protein basis. Using this assay, we assess: 1) what fraction of the proteome is degraded through autophagy upon a change in growth conditions; 2) what are the relative contributions of bulk and selective autophagy to this degradation; and 3) whether the degradation of some substrates is prioritized relative to that of others. Using the methylotrophic yeast P. pastoris, we find that enormous swaths of the proteome are degraded in an Atg9- and Atg11-dependent fashion as the microbe transitions between growth on different carbon sources. This result suggests that autophagy plays an outsized role in facilitating this nutrient adaptation, and our method directly identifies both the bulk and selective autophagic substrates targeted. Additionally, we observe that canonical selective autophagy substrates are degraded at different rates and in a growth-media dependent manner, suggesting a form of substrate prioritization. In sum, our study establishes a novel autophagy assay that could be readily extended to other model organisms, enumerates autophagic substrates in the yeast P. pastoris, and highlights the relative contributions of bulk and selective autophagy to nutrient adaptation in this methylotrophic yeast.