Neuronal communication relies on neurotransmitter release from synaptic vesicles (SVs), whose dynamics are controlled by Ca2+-dependent pathways, as many thoroughly studied phosphorylation cascades. However, little is known about other post-translational modifications, as ubiquitination. To address this, we analysed resting and stimulated synaptosomes (isolated synapses) by quantitative mass spectrometry. We identified more than 5,000 ubiquitination sites on ~2,000 proteins, the majority of which participate in the SV recycling processes. Several proteins showed significant changes in ubiquitination in response to Ca2+ influx, with the most pronounced changes in CaMKIIα and the clathrin adaptor protein AP180. To validate this finding, we generated a CaMKIIα mutant lacking the ubiquitination target (K291), and analyse it in both neurons and non-neuronal cells. K291 ubiquitination influences CaMKIIα activity and synaptic function, by modulating its autophosphorylation at a functionally important site (T286). We suggest that activity-dependent ubiquitination is an important regulator of synaptic function.

Validation of selected K-GG modified peptides in isolated synapses: Based on the results from previous DDA acquisition experiments, we selected 54 precursors from 27 K-GG modified peptide, 9 of which showed significant changes in their abundance upon calcium influx in isolated synapses (synaptosomes). Stimulated-vs- rested synaptosomes were used for the validation experiments. Targeted MS measurements using Parallel Reaction Monitoring (PRM) were performed: a linear gradient ranging from 12% to 36% buffer B for 43 min followed by a linear increase to 45% buffer B for 3 min was used on an UltiMate-3000 RSLC nanosystem (Thermo Fisher Scientific, Waltham, USA) coupled to an Orbitrap Exploris 480 (Thermo Fisher Scientific, Bremen, Germany), which was operated in targeted mass acquisition mode switching between MS1 and targeted MS2 scans. Within a 3-second cycle time one MS1 scan in the range of 350-1300 m/z was acquired with a maximum injection time of 50 ms followed by MS2 spectra derived from the targeted peptides. Specifically, heavy and light peptides matching the m/z values defined in the precursor isolation list were isolated with a 1 m/z isolation window and fragmented with a normalised collision energy (NCE) of 28%. MS2 fragment spectra were acquired with a resolution of 60,000, an AGC target of 5x105 and a maximum injection time of 120 ms. PRM analysis of CaMKIIα in synaptosomes and HeLa cells: Based on previous experiments, we selected K-GG modified CaMKIIα peptide and phosphorylated CaMKIIα peptide that changes significantly upon calcium influx in isolated synapses, along with the doubly modified peptide species (phosphorylated and ubiquitinated). CaMKIIα peptides that were not found to be modified were additionally selected to monitor the overall CaMKIIα abundance in our samples. Targeted MS measurements using Parallel Reaction Monitoring (PRM) were performed as described above.

Validation of selected K-GG modified peptides in isolated synapses: Resting -vs- stimulated synaptosomes were mildly centrifuged at 6900 g for 15 s in a bench-top centrifuge. The resulting pellet was lysed in lysis buffer containing 6 M guanidine hydrochloride, 50 mM HEPES, pH 8, 10 mM TCEP, 40 mM CAA, 5 mM EDTA, 50 µM PR-619, 1x Halt protease and phosphatase inhibitor cocktail. Proteins were reduced and carbamidomethylated in the presence of 10 mM TCEP/40 mM CAA at 50 °C for 30 min. 2 mg of synaptosomal proteins were precipitated using the methanol/chloroform precipitation method and subsequently digested with LysC and trypsin using a protease-to-protein ratio (w/w) of 1:200 and 1:50, respectively. K-ε-GG peptide enrichment was performed using the K-ε-GG-specific antibody (PTM-Scan HS Ubiquitin/SUMO remnant motif K-ε-GG kit, Cell Signaling Technology, Kit#59322) covalently linked to magnetic beads. For the validation of selected ubiquitination sites 200 fmol of standard synthetic SpikeTideL K-ε-GG peptides (JPT Peptide Technologies, Berlin, Germany) were added to the endogenous peptide mixture before K-ε-GG peptide enrichment. For the absolute quantification, the peptide mixture was split into two equal aliquots of 1 mg each and 350 fmol of standard AQUA QuantPro peptides (Thermo Fisher Scientific, Waltham, USA) were added to each aliquot before K-ε-GG peptide enrichment. For the phosphatase treatment, dried K-ε-GG peptides were resuspended in 1x CutSmart buffer (New England Biolabs, B7204S) and incubated with 2.5 u of QuickCIP phosphatase (New England Biolabs, M0525L) at 37 °C for 1 h. After that, QuickCIP was heat inactivated at 80 °C for 2 min and the peptide solutions were desalted using C18 StageTips (Empore C18 SPE Disks, Sigma Aldrich, Darmstadt, Germany) prior to PRM-MS analysis. PRM analysis of CaMKIIα in HeLa cells: HeLa cells stably exressing CaMKIIα were stimulated with 2.5 µM of the calcium ionophore, ionomycin (Sigma Aldrich, Darmstadt, Germany) in the presence of 1.8 mM CaCl2 in a cell incubator at 37 °C, and 5% CO2 for 7 min. Then, the medium was discarded and the cells were washed one time with PBS (Company, Cat#), followed by addition of lysis buffer (0.5% v/v NP40, 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA; 50 µM PR-619, 40 mM CAA, 1x Halt protease and phosphatase inhibitor cocktail). Cells were scraped off and the cell lysates were transferred to tubes. Nuclei were pelleted by centrifuging at 10,000 x g for 30 s at 4°C in a bench-top centrifuge. The supernatants were transferred to new tubes and any remaining DNA was further digested by the Pierce universal nuclease (250 U) (Thermo Fisher Scientific, Waltham, USA) in the presence of 4 mM MgCl2 for 30 min at 37 °C. Subsequently, proteins were reduced and alkylated in the presence of 20 mM TCEP/40 mM CAA at 37 °C for 30 min. Afterwards, equal amounts of proteins as determined by BCA assay were cleaned up by the single-pot, solid-phase-enhanced sample-preparation (SP3) method. Standard synthetic AQUA peptides (Technologies, Thermo Fisher Scientific, Waltham, USA) were added to the endogenous peptide mixture and peptides were dried in a centrifugal Savant SpeedVac (Thermo Fisher Scientific, Waltham, USA). Dried peptides were used either for K-GG peptide enrichment or phospho-peptide enrichment prior to PRM-MS analysis.