Chemical Proteomics Reveals Protein Tyrosination Extends Beyond the Alpha‐Tubulins in Human Cells

Tubulin detyrosination‐tyrosination cycle regulates the stability of microtubules. With respect to α‐tubulins, the tyrosination level is maintained by a single tubulin‐tyrosine ligase (TTL). However, the precise dynamics and tubulin isoforms which undergo (de)tyrosination in neurons are unknown. Here, we exploit the substrate promiscuity of the TTL to introduce an O‐propargyl‐l‐tyrosine to neuroblastoma cells and neurons. Mass spectrometry‐based chemical proteomics in neuroblastoma cells using the O‐propargyl‐l‐tyrosine probe revealed previously discussed tyrosination of TUBA4A, MAPRE1, and other non‐tubulin proteins. This finding was further corroborated in differentiating neurons. Together we present the method for tubulin tyrosination profiling in living cells. Our results show that detyrosination‐tyrosination is not restricted to α‐tubulins with coded C‐terminal tyrosine and is thus involved in fine‐tuning of the tubulin and non‐tubulin proteins during neuronal differentiation.