Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods

Abstract

The antigenic specificity of an unusual antinuclear antibody pattern in three patient sera was identified after separating HeLa-cell nuclear extracts by two-dimensional (2D) gel electrophoresis and localizing the antigens by immunoblotting with patient serum. Protein spots were excised from the 2D gel and their contents were analyzed by matrix-assisted laser desorption-ionization (MALDI) or nanoelectrospray ionization time-of-flight (TOF) tandem mass spectrometry (MS) after in-gel digestion with trypsin. A database search identified the proteins as the C1 and C2 heterogeneous nuclear ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein-separation methods and mass-spectrometric peptide mapping in combination with database searches are powerful tools in the identification of novel autoantigen specificities.

Figure 1

Indirect immunofluorescence pattern of anti-C1/C2-positive patient serum on HEp-2 cells. Cells were incubated with diluted patient serum (1:160), and bound IgG was detected by fluorescence microscopy after incubation with fluoresceinylated rabbit anti-human IgG. Strongly positive, very regular, coarse nuclear staining that spares nucleoli is observed. Original magnification is 200×. Scale bar is 10 μm.

Figure 2

Immunoblotting using different patient sera (2-5) and a normal control serum (1) diluted 1:100 on blots of HeLa-cell nuclear extracts separated on SDS-PAGE. The serum examined in lane 2 is identical to the one shown in Fig. 1, and the serum in lane 5 is a sample from the original patient described by Stanek et al [4]. Developed with anti-IgG antibodies. Molecular masses (×10^-3) of standard proteins are indicated on the left.

Figure 3

Isolation of antibody-reactive proteins by 2D gel electrophoresis and immunoblotting. (a) Two-dimensional gel electrophoresis of HeLa-cell nuclear extract. Molecular-weight marker positions are indicated on the left. Isoelectric focusing was in the horizontal dimension with the anode on the left. The gel is silver-stained. (b) Immunoblot from a separation similar to the one shown in (a), developed with the patient serum diluted 1:200. (c) Two-dimensional electrophoresis of a fraction of the nuclear extract purified by reversed-phase HPLC. (d) The immunoblot of the separation in (c), developed with a 1:200 dilution of patient serum. The two prominent spots marked with a circle in (c) were cut out separately and analyzed by MS after in-gel trypsin digestion.

Figure 4

Identification of C1/C2 hnRNPs by MS and by tandem MS sequencing. (a) Collision-induced dissociation spectrum of a doubly charged ion at m/z 580.32 Da corresponding to the sequence 207-216 (VDSLLENLEK) of the C2 hnRNP. The Y ion signal series is shown together with the deduced sequence giving the peptide sequence tag (632.36)LLSD(1060.70). (b) Two sections of the MALDI-MS peptide-mass maps showing the difference between the upper (C2) hnRNP and the lower (C1) hnRNP spots excised from 2D gels (cf Fig. 3c). The signal at m/z 3433.6 Da detected in the trypsin-digested material from the upper spot could be identified as the tryptic peptide that includes the 13-amino-acid insert in C2 hnRNP (illustrated in the figure). Only the corresponding peptide without the insert could be detected (at m/z 2101.9 Da) in the peptide-mass map from the lower spot. Signals derived by autoproteolysis of trypsin are marked with stars.