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. Electronic supplementary material The online version of this article (10.1186/s12014-019-9246-0) contains supplementary material, which is available to authorized users.

Using phosphoproteomics and next generation sequencing to discover novel therapeutic targets in patient antibodies

“…Music and coworkers [54] developed an efficient immunemass spectrometry approach to identify serum autoantibodies. All IgG antibodies, including autoantibodies, in serum are captured and purified on protein G beads, to be followed by addition of a protein mix containing thousands of human proteins.…”

“…Then, the beads are washed extensively, followed by capture of antigen by antibodies that were digested by trypsin and subsequently identified by shotgun tandem MS analysis. This MS method can accurately quantify and identify proteins that may be difficult or even impossible to detect when other technology is used [54] (Figure 2).…”

“…Methodology to identify antigens and autoantibodies from capturing with Protein G coated beads by mass spectrometry. (Copyright, Music et al[54]). …”

Predicting response and toxicity to PD-1 inhibition using serum autoantibodies identified from immuno-mass spectrometry

“…Details are stated in Extended data 30 . A similar method was recently validated in a proof-of-concept study that included autoantibodies to CUB and zona pellucida-like domain-containing protein 1 and pancreatic secretory granule membrane major glycoprotein 2 in the sera of patients with inflammatory bowel disease 31 .…”

Correction to: A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

“…In the original version of the article [1], an error was noticed under the heading “Immunoprecipitation on protein G magnetic beads” in “Methods” section. The unit symbol in the text that reads as “> 27 µl human” should read as “> 27 µg human”.…”