Quantitative analysis of protein ubiquitination levels in wild-type and ubp5 heterozygous mutant maize kernels was performed using DDA (Data-Dependent Acquisition) proteomics

Protein extraction was performed from maize kernels of wild-type and ubp5 heterozygous mutants, collected 30 days after self-pollination. For quantifying ubiquitin levels within the enzymatic digests, a previously established method was adapted with certain modifications. Two milligrams of protein were subjected to alkylation reduction using the aforementioned technique. The reduced proteins were then enzymatically digested employing a trypsin/lys-C protease mix (A41007, Thermo) in 50 mM ammonium bicarbonate overnight. Subsequently, the resulting digest was purified using a MonoSpin C18 spin column (GL Sciences, 5010-21701), eluted with 60% acetonitrile, and rapidly dried under vacuum conditions.Peptide segments were reconstituted in IAP buffer [50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl], and the isolation of ubiquitin peptides was performed by precipitating them using a K-ɛ-GG-specific antibody (A20303, ABclonal) bound to protein A Dynabeads (10001D, Thermo). Elution of peptides was carried out using 0.15% (vol/vol) TFA, followed by desalting through a MonoSpin C18 spin column and concentration under vacuum using a concentrator.