Peptides from trypsin digestion of native proteomes reveal that size, charge and local sequence motifs influence cleavage speed.

Sotolon (4,5-dimethyl-3-hydroxy-2,5-dihydrofuran-2-one) is a powerful odorant usually pointed out as being responsible not only for the characteristic curry notes of the finest fortified wines but also for the off-flavour notes in prematurely oxidized white wines. Most methods reported in literature for quantifying sotolon in wines are quite laborious and use large volumes of organic solvents. Thus, in the present study, the development of a simple, fast, and environment-friendly method for the quantification of sotolon in fortified wine is herein presented. The proposed method uses a single-step liquid-liquid extraction followed by RP-LC-MS/MS and was optimized using a full factorial design. The method showed good linearity (R² = 0.9999), intra- and interday precision lower than 10% RSD, recovery of about 95%, and high sensitivity (LOQ of 0.04 μg/L). The method was applied to analyse 44 fortified wines from different styles (from dry to sweet wines) and ages (3–115 years old), and it was found that it covers the concentration range usually found for this compound in this kind of alcoholic beverages, which was found to be within 6.3–810 μg/L. Thus, it can be concluded that this method can be used as an accurate tool for the rapid analysis of sotolon, since the early stages of its formation up to long ageing periods.

Madeira wine (MW) oxidative aging results in the formation of several key aromas. Little is still known about their odor relevance to the aroma of the most commercialized MWs. This report presents an in-depth study of the odor impact of sotolon in MW blends. First, its odor perception was estimated in MWs according to ASTM E679, testing different 3-year-old (3-yo) commercial blends. The odor relevance of sotolon in the aroma of 3-, 5-, and 10-yo commercial blends (89 MWs) was then appraised by calculating its Odor Activity Value (OAV), after determining its content by RP-HPLC-MS/MS. The sotolon odor perception in MW was as low as 23 µg/L, although it was found that little differences in the wine matrix influenced its perception. OAVs varied between 0.1 and 22, increasing with the blend age. Considering that 16% of the OAVs are higher than 10 (mostly ≥ 10-yo), sotolon was found to be a key contributor to the overall aroma MW blends.

Trypsin is a serine protease that plays a pivotal role in protein digestion, being extensively used in various proteomics workflows due to its cleavage profile and specificity. Understanding trypsin’s enzymatic behavior is thus critical. We have employed Above-Filter Digestion Proteomics (AFDIP) to investigate trypsin’s cleavage preferences in HeLa cell lysates, preserving the proteins’ native state. We quantified over 18,000 unique peptides and correlated their emergence rates with cleavage window sequence motifs and physicochemical properties. Contrary to previous studies performed on denatured proteomes, we found that in native proteins cleavages at lysine residues were more abundant and faster than at arginine residues, and that physicochemical properties of the peptides affected their emergence times. These findings may need to be taken into account when interpreting the results of limited proteolysis experiments as well as when designing a food protein with extremely fast digestion times.