Rationale: Investigation of non-covalent complexes of proteins using Affinity Mass Spectrometry (AMS) represents a major challenge in modern biomedical research. However, many experimental obstacles can make AMS data analysis complex. Additionally, sample purity and size of the protein may still pose significant challenges.
Methods: Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) was used for initial mapping of protein samples. nanoESI (electrospray ionization) quadrupole-time-of-flight (QTOF) MS was used for mapping of protein samples under native conditions and subsequent AMS studies. The human galectin-3 protein sample was expressed in E. coli.
Results: Full length galectin-3 was difficult to work with, due to several truncated forms observed after the purification procedures. On the other hand, galectin-3C produced excellent quality nanoESI-MS spectra. A covalent adduct of lactose was found to be located on residue Lys 176. Functional AMS control studies indicated that galectin-3 interactions with oligosaccharides may be dependent on its charge.
Conclusions: Mass spectrometry represents a valuable tool that can be efficiently used for structural characterization of protein samples prior to functional analyses. By means of accurate mass measurements, many protein truncations can be identified based on mass alone. Analysis of covalent adducts is more challenging. Finally, for AMS studies, careful use of controls may reveal charge-dependence of protein-oligosaccharide interactions. Copyright © 2016 John Wiley & Sons, Ltd.
Copyright © 2016 John Wiley & Sons, Ltd.