From Grapevine Tissues to Proteome Coverage: Analytical Workflows for Protein Extraction, Fractionation and Mass Spectrometry

Grapevine (Vitis spp.) proteomics is an important tool for investigating berry development, stress responses, dormancy, root adaptation and extracellular defense processes. However, many grapevine tissues and extracellular matrices remain analytically challenging because of relatively low recoverable protein abundance and high levels of interfering compounds, including polyphenols, polysaccharides, sugars, pigments, lipids and structural polymers. This review provides a workflow-oriented overview of analytical strategies used in grapevine proteomics, covering protein extraction and clean-up, fractionation, digestion, mass spectrometry and bioinformatic processing. Matrix-adapted approaches based on TCA/acetone precipitation, phenol extraction and SDS-, urea- or guanidine-containing buffers are considered across major grapevine tissues and extracellular matrices. Gel-based, GeLC and gel-free LC–MS/MS workflows, peptide fractionation, label-free and isobaric quantification, data-independent acquisition (DIA) and post-translational modification (PTM)-oriented strategies are critically compared, with emphasis on their effects on protein recovery, proteome coverage, reproducibility and cross-study comparability. Despite advances in high-resolution LC–MS/MS, fully gel-free bottom-up workflows remain less frequently represented than gel-assisted or protein-level-prefractionated approaches. Future priorities include improved matrix-specific clean-up, workflow standardization, reference materials and quality-control procedures, and more transparent reporting of analytical and bioinformatic parameters.