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Interaction of β-Lactoglobulin with Small Hydrophobic Ligands - Influence of Covalent AITC Modification on β-LG Tryptic Cleavage

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Abstract

Covalent modification of proteins with bioactive organosulfur compounds is suggested to reduce the smell and increase the stability of the compound. Aside from these effects the covalent modification may also alter the physiochemical properties of the protein. In this study, the whey protein β-lactoglobulin (β-LG) was covalently modified with bioactive organosulfur compound allyl isothiocyanate (AITC), originating from cabbage. Native and AITC modified β-LG were subjected to tryptic and chymotryptic digestion to assess the influence of the covalent modification on peptide formation. AITC was shown to modify at least 13 different amino acid residues, containing thiol- or amino groups, in a concentration dependent manner. Therefore, AITC modification can be controlled to some extent. Besides cysteine thiols, the most accessible amino groups for AITC modification were found at the N-terminal end of the protein (residues L1 and K8) along with lysine residues K91, K77 and K83. Higher amount of AITC addition resulted in a significant blocking of several tryptic cleavage sites of β-LG (for example residue K14) resulting in longer peptides, influencing the concentration of certain bioactive peptides following tryptic cleavage.

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Abbreviations

ACE:

Angiotensin-Converting-Enzyme

ACN:

Acetonitrile

AITC:

Allyl isothiocyanate

ANOVA:

Analysis of Variance’

β-LG:

β-lactoglobulin

CID:

Collision-induced dissociation

FA:

Formic acid

FT:

Fourier Transformation

GC:

Gas chromatography

HCD:

Higher energy collision dissociation

HEPES:

2-(4-(2-hydroxyethyl)- 1-piperazinyl)-ethanesulfonicacid

LC-ESI-MS:

Liquid chromatography-electrospray ionization-mass spectrometry

LFQ:

Label free quantification

MS:

Mass spectrometry

m/z:

Mass/charge

NF:

Normalization factor

rH:

Hydrodynamic radius

PEG:

Polyethylene glycol

RSH:

Reactive sulfhydryl groups

RT:

Retention Time

TFA:

Trifluoroacetic acid

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Acknowledgments

This project was funded by the BMBF program FOCUS, TP 5.3 (ProteoMod) and TP 3.4 (LactoTrans) and SFB877 (Project Z2).

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Authors and Affiliations

  1. Christian-Albrechts-Universität zu Kiel, Kiel, Germany

    Julia Katharina Keppler, Tomas Koudelka, Kalpana Palani, Andreas Tholey & Karin Schwarz

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  1. Julia Katharina Keppler
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Correspondence to Julia Katharina Keppler.

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Table 1

Peptides identified in β-LG native or modified with different concentrations of AITC by LC-ESI-MS2 after tryptic digestion. All modified amino acids are marked in bold with an asterisk. Where modified amino acid could not be identified unambiguously, the whole peptide sequence is marked in brackets and an asterisk. Only those peptides that were identified in at least two of the three biological replicates are listed. A.U. = Arbitrary Units, No = Number, RT = retention time, SEM = Standard error mean. (DOCX 38 kb)

Table 2

Peptides identified in β-LG native or modified with different concentrations of AITC by LC-ESI-MS2 after chymotryptic digestion. Modified amino acids are marked in bold and with an asterisk. Only those peptides that were identified in at least two of the three biological replicates were listed. Where modified amino acid could not be identified unambiguously, the whole peptide sequence is marked in brackets and an asterisk. A.U. = Arbitrary Units, No = Number, RT = retention time, SEM = Standard error mean. (DOCX 86 kb)

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Keppler, J.K., Koudelka, T., Palani, K. et al. Interaction of β-Lactoglobulin with Small Hydrophobic Ligands - Influence of Covalent AITC Modification on β-LG Tryptic Cleavage. Food Biophysics 9, 349–358 (2014). https://doi.org/10.1007/s11483-014-9361-4

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