Ubiquitin-like proteins (UBLs) such as SUMO, Nedd8 or UFM1 are conjugated to target proteins to regulate their activity, stability, subcellular localization, or macromolecular interactions. In recent years, UBLs have become an important subject of study in biomedical research as they play a role in central biological processes like stress response, and as UBL dysfunction is associated with many pathological states, including neurodevelopmental and neurodegenerative disorders. Knowledge of the modified lysine residues within a conjugated substrate is key for a detailed functional analysis, e.g. by replacing acceptor lysine with non-modifiable arginine residues. However, despite advances in mass spectrometry (MS)-based proteomics, mapping of UBL modification sites remains difficult not only because of the low stoichiometry of conjugation. Particular technical challenges include the detection and sequencing of low-abundant branched peptides, in which the size of the remnant from the UBL may vary considerably depending on the type of UBL present and the protease used for digestion during sample preparation.

To screen for substrates conjugated with SUMO2, the most abundant SUMO paralogue in neurons, we made use of a novel HA-SUMO2 knock-in mouse model, which expresses HA-tagged SUMO2 from the endogenous genetic locus. Using anti-HA affinity purification from whole brain, followed by label-free protein quantification, we identified several hundred putative SUMO2 candidates, including the transcription factor Zbtb20, of which we have previously characterized the SUMO acceptor sites [1]. We thus selected HEK cells expressing both tagged Zbtb20 and tagged SUMO2 as model for establishing a proteomic workflow for UBL conjugation site mapping. Partially using the strategy introduced by Hendriks et al. [2] as a starting point, we revisited critical steps of the workflow and established optimized elution conditions of the anti-tag affinity purification, C8 stagetip-based enrichment of branched peptides, and MS acquisition parameters with customized mass cutoffs and collision energies on an Orbitrap Exploris 480 instrument. Our workflow will be used in future for the site mapping of synaptic UBL targets with the aim of gaining insights into the role of UBL alterations in synapse biology and pathology.

[1] Ripamonti S, Shomroni O, Rhee JS, Chowdhury K, Jahn O, Hellmann KP, Bonn S, Brose N, Tirard M. SUMOylation controls the neurodevelopmental function of the transcription factor Zbtb20. J Neurochem 2020, 154:647.

[2] Hendriks IA, Lyon D, Su D, Skotte NH, Daniel JA, Jensen LJ, Nielsen ML. Site-specific characterization of endogenous SUMOylation across species and organs. Nat Commun 2018, 9:2456.