A.M.E.J.).SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion and contribute to homoeostasis, pathogen defense, cell expansion, and growth in plants. In Arabidopsis (Arabidopsis thaliana), two homologous Qa-SNAREs, SYNTAXIN OF PLANTS121 (SYP121) and SYP122, facilitate the majority of secretory traffic to the plasma membrane, and the single mutants are indistinguishable from wild-type plants in the absence of stress, implying a redundancy in their functions. Nonetheless, several studies suggest differences among the secretory cargo of these SNAREs. To address this issue, we conducted an analysis of the proteins secreted by cultured wildtype, syp121, and syp122 mutant Arabidopsis seedlings. Here, we report that a number of cargo proteins were associated differentially with traffic mediated by SYP121 and SYP122. The data also indicated important overlaps between the SNAREs. Therefore, we conclude that the two Qa-SNAREs mediate distinct but complementary secretory pathways during vegetative plant growth.

The in‐gel digestion of proteins for analysis by liquid chromatograph mass spectrometry has been used since the early 1990s. Although several improvements have contributed to increasing the quality of the data obtained, many recent publications still use sub‐optimal approaches. Updates of the in‐gel digestion protocol has been presented in the study. It has been shown that alternative reducing, alkylating agent reactions, and tryptic digestion buffers increase peptide and protein identification and reduce incubation times. The results indicate that a simultaneous and short, high temperature reduction and alkylation reaction using Tris(2‐carboxyethyl)phosphine hydrochloride and chloroacetamide with a subsequent gel wash improve protein identification and sequence coverage, and diminish peptide side reactions. Additionally, use of 4‐(2‐hydroxyethyl)piperazine‐1‐ethanesulfonic acid buffer allows a significant reduction in the digestion time improving trypsin performance and increasing the peptide recovery. The updates of the in‐gel digestion protocol described here are efficient and offer flexibility to be incorporated in any proteomic laboratory.