Post-translational modifications drive protein stability to control the dynamic beer brewing proteome

Abstract

Mashing is a key step in beer brewing in which starch and proteins are solubilized from malted barley in a hot water extraction and digested to oligomaltose and free amino nitrogen. We used SWATH-MS to measure the abundance and site-specific modifications of proteins throughout a small-scale pale ale mash. Proteins extracted from the malt at low mash temperatures decreased precipitously in abundance at higher temperatures due to temperature-induced unfolding and aggregation.Correlation analysis of temperature-dependent abundance showed that sequence and structure were the main features that controlled protein abundance profiles. Partial proteolysis by barley proteases was common early in the mash. The resulting proteolytically clipped proteins were particularly temperature sensitive and were preferentially lost at high mash temperatures, while intact proteins remained soluble.

Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

“…The protein intensity output from PeakView was recalculated by eliminating the value of peptides measured with a FDR > 0.01 ( Supplementary Table S5), using a modified version of our previously described script [63] (Supplementary information). To calculate the relative changes in protein abundance through time in the bioreactor, the protein abundance data for each protein in each day was normalized to the abundance of trypsin in that sample, as previously described [47] ( Supplementary Table S5). The mean (N=3) for each trypsin-normalized protein abundance at each time point was log10 transformed and plotted as a heatmap using GraphPad Prism v7.0a and 8.2.0 for Mac OS X (GraphPad Software, San Diego, California USA).…”

“…In DIA, all peptides eluting across a liquid chromatography (LC) gradient are fragmented according to pre-determined size (mass/charge) windows. By summing the abundance of pre-selected fragment ions of interest present in specific windows at specific retention times, it is possible to calculate the stoichiometry of modification and the relative abundance of differently post-translationally modified peptide variants and proteins in each sample [45][46][47][48][49][50][51]. DIA is superior to data dependent acquisition (DDA) workflows in that DDA fragmentation is intrinsically biased towards the more abundant peptides, while DIA allows measurement of all detectable analytes, allowing for a full exploration of the precursor landscape in a sample [51,52].…”

Quantitative data independent acquisition glycoproteomics of sparkling wine

“…Using this approach, we investigated the effects of aging on lees and the use of different yeast strains during the second fermentation on the molecular complexity and composition of sparkling wine. Acquisition of all THeoretical mass spectra (SWATH)-MS was performed using MSstats (v2.4) in R (32) with a significance threshold of P = 10 -5 as described previously (33,34). Sitespecificity of post-translational modifications was not a requirement for this study.…”

Increasing protein content and digestibility in sorghum grain with a synthetic biology approach

“…The ground sorghum seed samples were denatured/reduced/alkylated essentially as previously described (Peak et al 2016;Kerr and Schulz 2018). Briefly, 10 mg of cracked seed was resuspended in 300 µL 6 M guanidine HCl, 50 mM Tris HCl buffer pH 8, and 10 mM DTT and incubated with shaking at 30 ˚C for 30 min to solubilize and denature proteins, and to reduce disulfide bonds.…”