Multidimensional Analysis of Protease Substrates and Their Cellular Origins in Mixed Secretomes from Multiple Cell Types.

Abstract

Although extracellular proteases are confronted with substrate proteins expressed by multiple cell types in vivo, in most protease substrate discovery approaches, the test protease is exposed to a test proteome (secretome) derived only from a single cell type. This limits the potential substrate space and prohibits the formation of protein complexes constituted of components derived from multiple cellular origins. Mixing of secretomes collected from multiple cell types addresses this issue, but information on the cellular origin of a substrate protein is lost. Here, we describe a protocol and the corresponding data analysis workflow for a multidimensional substrate discovery approach termed SILAC -iTRAQ -TAILS that is based on hyperplexed terminal amine isotopic labeling of substrates (TAILS ), allowing identification of substrates and concomitant assignment to cellular origins in mixed secretomes within the same experiment.