Precision fermentation as a route to modify β-lactoglobulin structure through substitution of specific cysteine residues

Edman Degradation Reveals Unequivocal Analysis of the Disulfide Connectivity in Peptides and Proteins

“…This confirmed the expected pattern for disulfide bridges known for μ-KIIIA. , As compared to tandem mass spectrometric analysis, especially direct and fast assignment as well as the continuation of sequencing beyond a disulfide-bridged cysteine present a huge advantage for the analysis of this compound by ED. In contrast, in tandem MS disulfide bridges of μ-KIIIA lead to abrogation of fragmentation and to a significant drop in intensities of fragment ions beyond that cysteine within the bridge (Figure S4) as was also observed in previous studies. , This was especially aggravated by the vicinal cysteines in both terminals of μ-KIIIA that prevented sufficient assignment of fragment ion peaks in the oxidized form (Figure S4A). Thus, only analysis by ED would reveal the correct and completely assigned disulfide pattern for this peptide after partial reduction without any ambiguities and without great effort.…”

“…In contrast, in tandem MS disulfide bridges of μ-KIIIA lead to abrogation of fragmentation and to a significant drop in intensities of fragment ions beyond that cysteine within the bridge (Figure S4) as was also observed in previous studies. 21,24 This was especially aggravated by the vicinal cysteines in both terminals of μ-KIIIA that prevented sufficient assignment of fragment ion peaks in the oxidized form (Figure S4A). Thus, only analysis by ED would reveal the correct and completely assigned disulfide pattern for this peptide after partial reduction without any ambiguities and without great effort.…”

“…MS is predominantly used for peptide mapping and identification of cysteine residues and disulfide bonds after enzymatic digestion, reduction, and derivatization . MS, however, has limitations in determining the proper disulfide connectivity when suitable cleavage sites of digestive enzymes such as Arg/Lys for trypsin, Phe/Tyr/Trp for chymotrypsin, and Ser/Ala/Val for elastase are absent. , Other approaches, which do not require enzymatic digestion such as online electrochemical cells connected to MS, exist and were also reported to be used for disulfide bridge elucidation. − Recent improvements in MS fragmentation and detection approaches such as ion mobility and trapped ion mobility as well as in source decay were developed and also involved in disulfide bridge identification, especially for vicinal cysteines. − The elucidation of the disulfide connectivity then requires an accurate evaluation of the fragment pattern and the contained disulfide bonds. ,, In addition, MS data evaluation of complex samples or unknown sequences could be a difficult task due to the high number of fragments that may require specific expertise and extensive time for correct evaluation. ,− The loss of significant fragments being involved in a disulfide bridge, especially in case of low concentrated samples, can dramatically affect the final assessment. − Finally, although MS is a widely used tool for elucidating posttranslational modifications, it may not provide adequate differentiation between all possible isomers of a multiple disulfide-bonded peptide. ,,,, Therefore, all analytical methods available should be exhausted to determine the correct disulfide connectivity and also to provide quantitative information as required. Edman degradation (ED), which was introduced in the 1950s, is a method developed for the analysis of the primary amino acid sequence of a peptide (aa n ) (for a comparison between tandem MS and ED regarding the determination of the disulfide connectivity in disulfide-rich peptides and proteins see Table S1).…”

Dairy, Plant, and Novel Proteins: Scientific and Technological Aspects

“…However, bovine whey protein is about 52% β-lactoglobulin, 17% α-lactalbumin, 12% glycomacropeptides, 10% immunoglobulins, 5% serum albumin, 1.5% lactoferrin, and 2.5% other proteins [207]. The single precision fermentation derived β-lactoglobulin does not have the same amino acid profile and diverse protein structure, and thus it does not have an identical protein quality (PDCAAS and DIAAS values) as or similar functional properties to bovine milk proteins or whey proteins [205,229]. Brune et al [229] investigated substituting cysteine with alanine on the protein structure of precision fermentation-derived β-lactoglobulin to improve its functionality.…”

“…The single precision fermentation derived β-lactoglobulin does not have the same amino acid profile and diverse protein structure, and thus it does not have an identical protein quality (PDCAAS and DIAAS values) as or similar functional properties to bovine milk proteins or whey proteins [205,229]. Brune et al [229] investigated substituting cysteine with alanine on the protein structure of precision fermentation-derived β-lactoglobulin to improve its functionality. More research can be conducted to improve the functionality of fermentation-derived β-lactoglobulin, but it would be hard to compete with bovine dairy proteins, which have diverse protein components and structures that contribute to superior functionalities.…”