Updated project metadata. Small ubiquitin-like modifier (SUMO) typically conjugates to target proteins through isopeptide linkage to the ε-amino group of lysine residues. This posttranslational modification (PTM) plays pivotal roles in modulating protein function. As a key regulator of actin cytoskeleton dynamics, cofilins (CFLs) are well-known to undergo several different kinds of PTMs. Using mass spectrometry, our study identified the N-terminal α-amino group, rather than any of the ε-amino groups of the internal lysines, as the sole SUMO-conjugation site of cofilin-1 (CFL1).