The development of new approaches is often challenging, but is also critical to gain further insights that cannot be accessed by existing methods or technologies. In this study, we borrowed a technique initially developed by the group of Alice Ting for studies in eukaryotic cells and mitochondria. This technique is based on the covalent biotinylation of proteins at the proximity of an engineered variant of the soybean ascorbate peroxidase, called APEX2. Biotinylated proteins can be then enriched and identified by mass spectrometry. We adapted this technique in Escherichia coli cells in order to identify partners of the highly dynamic TssA protein that is involved in the different stages of the assembly of the Type VI secretion system (T6SS). The T6SS is a fascinating machine widespread in Gram-negative bacteria. It assembles a spring-like structure that can be compared to a crossbow or speargun, and used to inject effectors into target cells upon contraction. The APEX2 approach did not only allow to provide further insights on the assembly pathway of this multiprotein apparatus but also revealed a new player in T6SS that acts as a latch for the nano-crossbow.