Catalin Koro, Ewa Bielecka, Anders Dahl-Knudsen, Jan J. Enghild,. Carsten Scavenius, Johan G. Brun, Veronika Binder, Annelie Hellvard,. Brith Bergum ...
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European Journal of Immunology Supporting Information for DOI 10.1002/eji.201444869 Catalin Koro, Ewa Bielecka, Anders Dahl-Knudsen, Jan J. Enghild, Carsten Scavenius, Johan G. Brun, Veronika Binder, Annelie Hellvard, Brith Bergum, Roland Jonsson, Jan Potempa, Anna M. Blom and Piotr Mydel Carbamylation of immunoglobulin abrogates activation of the classical complement pathway
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eji-journal.eu
Supplementary materials
Sample Patient 1
Peptides
Sequence
sequenced
coverage
83
93%
Carbamylated peptide ALPAPIEKTISK
Site 334
Times
CID evidence
measured
ions
4
y5, y6, y6*, y7, y70, y8, y8*, y8*++, y9, y9*, y9++, y10, y100
VSNKALPAPIEKTIS
334
2
K
y5, y6, y6*, y7, y7*, y8, y8*, y8++, y10, y10*, y15++, b14++
Patient 2
93
94%
ALPAPIEKTISK
334
2
y5, y5*, y6, y6*, y6*++, y60, y7, y7*, y8, y8*, y80, y9, y9*, y9++, y10, y10*, y100, b8*
KPKDTLMISR
246
1
b9++
Supplementary Table 1. Lysine carbamylations sites in patient synovial fluid. Lysine carbamylations sites in patient synovial fluid detected using collision-induced fragmentation (CID) MS/MS. The sites are confirmed by manual review of CID fragmentation spectra verifying whether peak patterns are best explained by the presence of a lysine carbamylation. The listed y-ions (C-terminal fragments) and bions (N-terminal fragments) are the observed spectral evidence peaks confirming the assigned carbamylation. All of the identified peptides have mass deviations below 23ppm and the average deviation is 11.3ppm. Peptides sequenced indicate the number of IgG1 peptide spectra recorded for the sample, and sequence coverage indicates the percentage of the IgG1 sequence covered by the detected peptides.* Loss of NH3; 0 Loss of H2O; ++ Double charged ions.
Flow cytometric gating CDC
ADCC
Overlay Blue - Raji cells Red - NK cells
Supplementary Figure 1. Gating strategy. (A) Complement dependent cytotoxicity assay and (B) antibody-dependent cellular cytotoxicity assay.
