title: Application of amide hydrogen/deuterium exchange mass spectrometry for epitope mapping in human cystatin C.
abstract: Human cystatin C (hCC) is a small cysteine protease inhibitor whose oligomerization by propagated domain swapping is linked to certain neurological disorders. One of the ways to prevent hCC dimerization and fibrillogenesis is to enable its interaction with a proper antibody. Herein, the sites of interaction of hCC with dimer-preventing mouse monoclonal anti-hCC antibodies Cyst28 are studied and compared with the binding sites found for mAb Cyst10 that has almost no effect on hCC dimerization. In addition, hCC epitopes in complexes with native polyclonal antibodies extracted from human serum were studied. The results obtained with hydrogen-deuterium exchange mass spectrometry (HDX MS) were compared with the previous findings made using the excision/extraction MS approach. The main results from the two complementary MS-based approaches are found to be in agreement with each other, with some differences being attributed to the specificity of each method. The findings of the current studies may be important for future design of hCC dimerization inhibitors.
authors: Faculty of Chemistry, Department of Biomedical Chemistry, University of Gdańsk, Wita Stwosza 63, 80-952, Gdańsk, Poland.
authors: Martyna
authors: M
authors: Prądzińska
authors: Faculty of Chemistry, Department of Biomedical Chemistry, University of Gdańsk, Wita Stwosza 63, 80-952, Gdańsk, Poland.
authors: Izabela
authors: I
authors: Behrendt
authors: Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, S-171 77, Stockholm, Sweden.|Biomotif AB, 18212, Stockholm, Sweden.
authors: Juan
authors: J
authors: Astorga-Wells
authors: Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, S-171 77, Stockholm, Sweden.
authors: Aleksandr
authors: A
authors: Manoilov
authors: Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, S-171 77, Stockholm, Sweden. roman.zubarev@ki.se.
authors: Roman A
authors: RA
authors: Zubarev
authors: Faculty of Chemistry, Department of Biomedical Chemistry, University of Gdańsk, Wita Stwosza 63, 80-952, Gdańsk, Poland.
authors: Aleksandra S
authors: AS
authors: Kołodziejczyk
authors: Faculty of Chemistry, Department of Biomedical Chemistry, University of Gdańsk, Wita Stwosza 63, 80-952, Gdańsk, Poland.
authors: Sylwia
authors: S
authors: Rodziewicz-Motowidło
authors: Intercollegiate Faculty of Biotechnology, University of Gdańsk-Medical University of Gdańsk, Kładki 24, 80-822, Gdańsk, Poland. pczaplewska@gmail.com.
authors: Paulina
authors: P
authors: Czaplewska
mesh_terms: D000577:Amides / Q000737:chemistry / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D001665:Binding Sites; D055316:Cystatin C / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology*; D041961:Deuterium Exchange Measurement; D018604:Epitope Mapping; D006801:Humans; D051379:Mice; D055503:Protein Multimerization / Q000187:drug effects / Q000276:immunology
chemical_list: D000577:Amides; D000911:Antibodies, Monoclonal; D055316:Cystatin C
keywords: Epitope identification; HDX exchange; Human cystatin C; Mass spectrometry