title: Preliminary mass spectrometry characterization studies of galectin-3 samples, prior to carbohydrate-binding studies using Affinity mass spectrometry.
abstract: BACKGROUND
  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.
authors: Institute of Medical Physics and Biophysics, University of Münster, Robert-Koch-Strasse 31, D-48149, Münster, Germany.|Department of Biotechnology, University of Rijeka, Radmile Matejčić 2, 51 000, Rijeka, Croatia.
authors: Marko
authors: http://orcid.org/0000-0002-8387-2271
authors: M
authors: Jovanović
authors: Institute of Medical Physics and Biophysics, University of Münster, Robert-Koch-Strasse 31, D-48149, Münster, Germany.
authors: Jasna
authors: J
authors: Peter-Katalinić
mesh_terms: D001798:Blood Proteins; D004926:Escherichia coli; D037502:Galectin 3 / Q000032:analysis* / Q000737:chemistry*; D037161:Galectins; D006801:Humans; D007785:Lactose / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008970:Molecular Weight; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D001798:Blood Proteins; D037502:Galectin 3; D037161:Galectins; C428445:LGALS3 protein, human; D011994:Recombinant Proteins; D007785:Lactose; D008239:Lysine