A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

Abstract

Background: Autoantibodies are produced when tolerance to self-antigens is broken and they can be mediators of tissue injury and systemic inflammation. They are excellent biomarkers because they are minimally invasive to screen and are highly abundant in serum due to limited proteolysis and slow clearance. Conventionally used methods of identifying autoantibodies in patient sera include indirect immunofluorescence, enzyme-linked immunoabsorbent assays (ELISAs) and protein microarrays. Here we present a novel proteome-wide immuno-mass spectrometric method to identify serum autoantibody targets.

Methods: Serum samples from patients with inflammatory bowel disease (IBD) were analyzed by ELISA for the presence of autoantibodies to CUB and zona pellucida-like domain-containing protein 1 (CUZD1). Protein was extracted from the human pancreas as well as 16 other human tissues to make a complex tissue lysate protein mixture. Antibodies in patient sera were immobilized and purified on protein G magnetic beads and subsequently incubated with pancreatic lysate containing CUZD1 or the aforementioned complex tissue lysate. After extensive washing, antibody-bound protein antigens were trypsin-digested and identified using shotgun mass spectrometry.

Results: The protocol was optimized for the immunoaffinity purification of autoantibody targets from tissue lysate, using CUZD1 from pancreatic lysate and anti-CUZD1 autoantibodies present in IBD patient serum as a proof-of-concept. Pancreatic secretory granule membrane major glycoprotein 2, whose autoantibodies are a known biomarker of Crohn's disease, was also immunoprecipitated from IBD patient serum, as an additional internal positive control.

Conclusions: This study demonstrates the effectiveness of a proteomic approach to identify serum autoantibody targets, using immunoaffinity purification followed by tandem mass spectrometry. Our methodology is applicable for proteome-wide analysis of autoantibody targets in a wide variety of clinical settings.

Keywords: Autoantibodies; Biomarkers; Immuno-MS; Immunoprecipitation; Mass spectrometry; Protein G magnetic beads; Proteomics.

Fig. 1

Methodology used to identify anti-CUZD1 serum autoantibodies with protein G magnetic beads. In the first step, serum is added to the beads, to capture serum antibodies, some of which are anti-CUZD1 autoantibodies (blue color). After washing, the beads are exposed to a protein mix, in this case pancreatic lysate or a more complex tissue lysate. In the third step, beads are washed and the captured proteins (including CUZD1; blue dots) are trypsin-digested before identification with tandem mass spectrometry. For more details see text

Fig. 2

ELISA assay for identifying anti-CUZD1 serum autoantibodies. The method is described in detail in reference 12. Two different sera (Serum #1 and Serum #2) were tested against a previously confirmed positive serum control as well as a negative serum control. The fluorescence signal from the blanks was removed from each result. The two sera had higher autoantibody titers than the previously identified positive control. These sera were used to develop our MS-based assay

Fig. 3

MS2 spectra of the peptide SYLEAFNSNGNNLQLK originating from CUZD1 protein, annotated with b and y ions

Fig. 4

MS2 spectra of peptide NWVSVTSPVQASAcr, originating from GP2 protein, annotated with b and y ions

Fig. 5

Tryptic peptides corresponding to CUZD1 that were identified by our immuno-MS approach. These peptides are highlighted on the entire CUZD1 protein sequence. *Note: The peptide EIFLEIDK (E198-K205) was also identified as a miscleaved EIFLEIDKQCK (E198-K208). **Note: The peptide SYLEAFNSNGNNLQLK (S289-K304) was also identified as a miscleaved SYLEAFNSNGNNLQLKDPTCRPK (S289-K311). ***Note: The sequence was obtained from UniProt

Fig. 6

Tryptic peptides corresponding to GP2 that were identified by our immuno-MS approach. These peptides are highlighted on the entire GP2 protein sequence. ***Note: The sequence was obtained from UniProt