PMID- 32886488
OWN - NLM
STAT- MEDLINE
DCOM- 20210216
LR  - 20231112
IS  - 1520-6882 (Electronic)
IS  - 0003-2700 (Print)
IS  - 0003-2700 (Linking)
VI  - 92
IP  - 19
DP  - 2020 Oct 6
TI  - Site-Specific Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of 
      Individual Healthy Donors.
PG  - 13172-13181
LID - 10.1021/acs.analchem.0c02342 [doi]
AB  - Fc gamma receptors (FcgammaRs) translate antigen recognition by immunoglobulin G 
      (IgG) into various immune responses. A better understanding of this key element 
      of immunity promises novel insights into mechanisms of (auto-/allo-)immune 
      diseases and more rationally designed antibody-based drugs. Glycosylation on both 
      IgG and FcgammaR impacts their interaction dramatically. Regarding FcgammaR glycosylation 
      profiling, major analytical challenges are associated with the presence of 
      multiple glycosylation sites in close proximity and large structural 
      heterogeneity. To address these challenges, we developed a straightforward and 
      comprehensive analytical methodology to map FcgammaRIIIb glycosylation in primary 
      human cells. After neutrophil isolation and immunoprecipitation, glycopeptides 
      containing a single site each were generated by a dual-protease in-gel digestion. 
      The complex mixture was resolved by liquid chromatography-tandem mass 
      spectrometry (LC-MS/MS) providing information on the level of individual donors. 
      In contrast to recently published alternatives for FcgammaRIIIb, we assessed its 
      site-specific glycosylation in a single LC-MS/MS run and simultaneously 
      determined the donor allotype. Studying FcgammaRIIIb derived from healthy donor 
      neutrophils, we observed profound differences as compared to the soluble variant 
      and the homologous FcgammaRIIIa on natural killer cells. This method will allow 
      assessment of differences in FcgammaRIII glycosylation between individuals, cell 
      types, subcellular locations, and pathophysiological conditions.
FAU - Wojcik, Iwona
AU  - Wojcik I
AUID- ORCID: 0000-0002-9365-1616
AD  - Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC 
      Leiden, The Netherlands.
AD  - Glycoscience Research Laboratory, Genos Ltd., Zagreb 10000, Croatia.
FAU - Senard, Thomas
AU  - Senard T
AUID- ORCID: 0000-0001-5951-4127
AD  - Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC 
      Leiden, The Netherlands.
FAU - de Graaf, Erik L
AU  - de Graaf EL
AD  - Department of Experimental Immunohematology, Sanquin Research, and Landsteiner 
      Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, 
      The Netherlands.
FAU - Janssen, George M C
AU  - Janssen GMC
AD  - Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC 
      Leiden, The Netherlands.
FAU - de Ru, Arnoud H
AU  - de Ru AH
AD  - Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC 
      Leiden, The Netherlands.
FAU - Mohammed, Yassene
AU  - Mohammed Y
AUID- ORCID: 0000-0003-3265-3332
AD  - Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC 
      Leiden, The Netherlands.
FAU - van Veelen, Peter A
AU  - van Veelen PA
AD  - Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC 
      Leiden, The Netherlands.
FAU - Vidarsson, Gestur
AU  - Vidarsson G
AD  - Department of Experimental Immunohematology, Sanquin Research, and Landsteiner 
      Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, 
      The Netherlands.
FAU - Wuhrer, Manfred
AU  - Wuhrer M
AUID- ORCID: 0000-0002-0814-4995
AD  - Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC 
      Leiden, The Netherlands.
FAU - Falck, David
AU  - Falck D
AUID- ORCID: 0000-0003-3908-2376
AD  - Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC 
      Leiden, The Netherlands.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20200922
PL  - United States
TA  - Anal Chem
JT  - Analytical chemistry
JID - 0370536
RN  - 0 (FCGR3B protein, human)
RN  - 0 (GPI-Linked Proteins)
RN  - 0 (Receptors, IgG)
SB  - IM
MH  - Chromatography, Liquid
MH  - GPI-Linked Proteins/analysis/immunology
MH  - Glycosylation
MH  - Healthy Volunteers
MH  - Humans
MH  - Neutrophils/*chemistry/cytology
MH  - *Protein Interaction Mapping
MH  - Receptors, IgG/analysis/*immunology
MH  - Tandem Mass Spectrometry
PMC - PMC7547861
COIS- The authors declare the following competing financial interest(s): I.W. is 
      employed by Genos Ltd. and E.L.G is employed by Pepscope Ltd. No potential 
      conflict of interest was reported by the remaining authors.
EDAT- 2020/09/05 06:00
MHDA- 2021/02/17 06:00
PMCR- 2020/10/10
CRDT- 2020/09/04 17:08
PHST- 2020/09/05 06:00 [pubmed]
PHST- 2021/02/17 06:00 [medline]
PHST- 2020/09/04 17:08 [entrez]
PHST- 2020/10/10 00:00 [pmc-release]
AID - 10.1021/acs.analchem.0c02342 [doi]
PST - ppublish
SO  - Anal Chem. 2020 Oct 6;92(19):13172-13181. doi: 10.1021/acs.analchem.0c02342. Epub 
      2020 Sep 22.