Structural analysis of the interleukin-8/glycosaminoglycan interactions by amide hydrogen/deuterium exchange mass spectrometry

Methods. 2015 Nov 1;89:45-53. doi: 10.1016/j.ymeth.2015.02.011. Epub 2015 Feb 26.


The recruitment of different chemokines and growth factors by glycosaminoglycans (GAGs) such as chondroitin sulfate or hyaluronan plays a critical role in wound healing processes. Thus, there is a special interest in the design of artificial extracellular matrices with improved properties concerning GAG interaction with common regulating proteins. In this study, amide hydrogen/deuterium (H/D) exchange mass spectrometry (HDX MS) combined with molecular modeling and docking experiments was used to obtain structural models of proinflammatory chemokine interleukin-8 (IL-8) in complex with hexameric chondroitin sulfate. Experiments on the intact protein showed a difference in deuterium labeling of IL-8 due to chondroitin sulfate binding. The extent of deuteration was reduced from 24% to 13% after 2 min exchange time, which corresponds to a reduced exchange of approximately 10 backbone amides. By local HDX MS experiments, H/D exchange information on the complete sequence of IL-8 could be obtained. A significantly reduced H/D exchange, especially of the C-terminal α-helical region comprising amino acids 70-77 and to the loop comprising amino acids 27-29 was observed in the presence of chondroitin sulfate. HDX MS data were used to model the IL-8/chondroitin sulfate complex. The binding interface of IL-8 and chondroitin sulfate determined this way correlated excellently with the corresponding NMR based atomistic model previously published. Our results demonstrate that HDX-MS in combination with molecular modeling is a valuable approach for the analysis of protein/GAG complexes at physiological pH, temperature, and salt concentration. The fact that HDX-MS requires only micrograms of protein and GAGs makes it a very promising technique to address protein-GAG interactions.

Keywords: Amide hydrogen/deuterium exchange; Chemokine; Chondroitin sulfate; Extracellular matrix; Interleukin-8; Protein/glycosaminoglycan interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amides / chemistry*
  • Amino Acid Sequence
  • Deuterium Exchange Measurement / methods*
  • Glycosaminoglycans / analysis*
  • Glycosaminoglycans / genetics
  • Glycosaminoglycans / metabolism
  • Humans
  • Hydrogen / chemistry*
  • Interleukin-8 / analysis*
  • Interleukin-8 / genetics
  • Interleukin-8 / metabolism
  • Molecular Sequence Data
  • Protein Binding
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*


  • Amides
  • CXCL8 protein, human
  • Glycosaminoglycans
  • Interleukin-8
  • Hydrogen