Fig. 1: Experimental workflow and validation of ACPA IgG1 Fab profiling.

a Experimental workflow of the anti-citrullinated protein antibody (ACPA) IgG1 Fab profiling approach, which consists of three steps: (1) autoantigen-specific tandem affinity purification of ACPA from individual patient plasma using each one column functionalized with the non-specific CArgP2 control peptide (depicted as Arg) and the citrullinated CCP2 peptide (depicted as Cit); (2) affinity capturing of IgG from purified ACPA and subsequent generation of ACPA IgG1 Fab fragments by on-bead digestion using the IgG1-specific protease IgdE; (3) LC-MS-based Fab profiling of collected ACPA IgG1 Fab fragments. In comparison, total plasma IgG1 Fab profiles are obtained by applying total plasma directly to IgG affinity capturing (steps 2 and 3; top). b–e Validation of the approach by application of ACPA-positive rheumatoid arthritis (RA) patient plasma (blue) and ACPA-negative healthy donor (HD) plasma (green). b Chromatogram recorded following elution of ACPA affinity purification. c ACPA IgG and anti-TT IgG reactivity of plasma, ACPA-depleted plasma and purified ACPA determined by ELISA. All samples were assessed one time with technical replicates (n = 2) at the same dilution. Results are depicted as mean and standard deviation. Individual data points are overlaid. d ACPA IgG1 Fab profiles collected upon LC-MS-based Fab profiling. Unique Fab molecules are depicted as peak at their mass. e ACPA IgG1 Fab profile collected upon repetitive ACPA IgG1 Fab profiling. Profiling was repeated n = 2 additional times. The profile of one exemplary replicate is shown. Fab molecules shared between both replicates (blue) and unique for the profile depicted (grey) are indicated. The degree of overlap between both replicates is indicated as a heatmap.