Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion

Abstract

Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.

Interactions between Muscle and Bone—Where Physics Meets Biology

“…While the former requires secretory signal peptide sequences, classical endocrine signaling requires the production of large secretory vesicles packed with preformed protein and the rapid release upon defined signals [50,51] ( Figure 2C). There is no evidence that mesenchymal tissues express the tools for classical regulated secretion; the list of tissue harboring this capacity comprises truly endocrine cells of all glands, exocrine cells, neurons, immune cells, and oocytes [66][67][68][69].…”

Optimized Proteome Reduction for Integrative Top–Down Proteomics

“…Although there have been previous reports assessing, to some extent, the use of reducing reagents in 2DE, there does not appear to be a direct assessment between different sample types treated with these reagents, either alone or in combination [ 27 , 35 , 37 , 38 , 46 , 51 , 52 ]. It is acknowledged that there will not be a ‘perfect’ one-size-fits-all method; however, the goal here was to establish an optimized general protocol to enhance 2DE resolution and thus a breadth of research supported by routine integrative top–down proteomic analyses [ 7 , 8 , 9 , 11 , 12 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 ]. The data clearly indicate that for native proteome extracts, reduction using 100 mM DTT + 5 mM TBP prior to sample rehydration into IPG strips is superior to the current ‘standard’ as well as to other reagents previously reported in the literature.…”

Proteomics—The State of the Field: The Definition and Analysis of Proteomes Should Be Based in Reality, Not Convenience

“…The challenge both for the first dimension of 2DE and for MS analyses has been the incompatibility of SDS, which is broadly considered the gold standard for proteoform extraction. Nonetheless, effective combinations of automated frozen disruption, detergents, and denaturing agents have enabled even the resolution of complete membrane proteomes by 2DE, despite long-held dogma suggesting this was not feasible [ 4 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 ]. Indeed, refined extraction and 2DE protocols have established quantitative proteome analysis consistent with SDS extraction [ 122 ].…”