Quantitative Data-Independent Acquisition Glycoproteomics of Sparkling Wine.

Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analyzed the glycoproteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyze sequential window acquisition of all theoretical mass spectra data-independent acquisition mass spectrometry data based on glycopeptides identified by Byonic (Protein Metrics; version 2.13.17). We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the liqueur de tirage for secondary fermentation. We found that aging a cuvée on lees for 24 months compared with 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. To our knowledge, this work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic sequential window acquisition of all theoretical mass spectra/data-independent acquisition workflow that is broadly applicable to other sample types.

Targeting the undruggable: emerging technologies in antibody delivery against intracellular targets.

INTRODUCTION: Monoclonal antibodies have been utilized in clinical and basic research for the treatment of various malignancies. Whilst all therapeutically approved monoclonal antibodies or fragments thereof are directed against cell-surface receptors or proteins of the human secretome, intracellular antigen targeting strategies still await translation into the clinic. This contradicts the notion of antibodies being the magic bullet concept as many cancer targets are out of reach. AREAS COVERED: This review provides a summary of intracellular translocation strategies that were successfully employed for antibody delivery in preclinical studies. Examples encompass a variety of different approaches such as polymeric and lipid-based nanoparticles (NP), biomimetics, bispecific antibody constructs, the use of cell-penetrating peptides, as well as various sophisticated combinations thereof. We will further discuss endosomal escape as the major bottleneck in functional intracellular transport and provide suggestions on how to overcome current challenges. EXPERT OPINION: Despite significant advances in protein delivery technologies, reports of highly efficient transport vehicles are sparse when systemically applied in vivo. Consequently, more detailed mechanistic studies are needed to identify and optimize the molecular 'Achilles heel' of individual methodologies. Ultimately, to target intracellular proteins that have been undruggable in the past, a combination of strategies may be required.