Fast and Accurate Disulfide Bridge Detection.

Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multibillion dollar industry. Essential here is the quality control assessment of critical attributes, such as sequence fidelity, proper folding, and posttranslational modifications. Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition, and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis, the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background, which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within 1 h, allowing for high-throughput, high-accuracy disulfide mapping.

Microwave-Assisted Acid Hydrolysis vs. Conventional Hydrolysis to Produce Sapogenin-Rich Products from Fenugreek Extracts.

The acid hydrolysis of saponins is commonly performed by conventional heating to produce sapogenin-rich products of bioactive interest, but alternative hydrolysis methods and their impact on bioactivity have been unexplored. We compared the conventional method with microwave-assisted acid hydrolysis (MAAH) of a commercial saponin-rich extract from a typical saponin source, fenugreek, focusing on the study of temperature (100, 120, 130, 140, 150 °C) and time (10, 20, 30, 40 min) of hydrolysis. The impact of these factors was assayed on both the sapogenin yield and the bioactivity of the hydrolyzed products, specifically their antioxidant and lipase inhibitory activities. The highest sapogenin content (34 g/100 g extract) was achieved by MAAH at 140 °C and 30 min, which was higher than conventional hydrolysis at both reference conditions (100 °C, 60 min, 24.6 g/100 g extract) and comparative conditions (140 °C, 30 min, 17 g/100 g extract) (p < 0.001). Typical steroid artifacts from sapogenins were observed in very small amounts, regardless of the method of hydrolysis. Antioxidant activity of MAAH hydrolyzed extracts (around 80% DPPH inhibition) was barely affected by time and temperature, but pancreatic lipase inhibitory activity was higher (>65%) at lower MAAH temperature (<130 °C) and time (<30 min) of hydrolysis. MAAH is shown as a valid alternative to produce selective sapogenin-rich extracts from fenugreek with minor impact on their bioactivities, and whose magnitude can be modulated by the hydrolysis conditions.