A proteomics-based method for identifying antigens within immune complexes

Abstract

A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.

Fig 1. Experimental workflow showing the strategy involved to recover and purify ICs.

Fig 2. Experimental workflow used to recover and purify each peak from the affinity purification step prior to protein sequencing.

Fig 3. H1N1 Western blotting of PEG precipitates from two healthy adult control samples, healthy adult sera, PBS as a negative control and flu vaccine as a positive control, showed that PEG can precipitate in vitro ICs.

Lane one is the Precision Plus protein standard ranging from 31 to 460 kDa. Three μg of protein per lane was loaded on a 3–8% Tris-acetate denaturing SDS-PAGE gel, blotted to nitrocellulose membrane and incubated with rabbit anti-H1N1 influenza A virus (primary antibody), goat anti-rabbit Immunoglobulin HRP (secondary antibody) and stained using the ECL kit. D stands for Donor, V for Vaccine and Negative control (Lane 7) is PEG with PBS. Lane 15 is a positive control, the influenza vaccine.

Fig 4. H1N1 Western blotting of healthy adult serum and serum spiked with vaccine, PEG precipitates from two healthy adult donor samples showed that influenza antigen was only detected in the PEG precipitated serum samples spiked with vaccine.

Lane 1 of the gel is the Precision Plus protein standard ranging from 31 to 460 kDa. Three μg of protein per lane was loaded on a 3–8% Tris-acetate denaturing SDS-PAGE gel, blotted to nitrocellulose membrane and incubated with rabbit anti-H1N1 influenza A virus (primary antibody), goat anti-rabbit immunoglobulin HRP (secondary antibody) and stained using the ECL kit. D stands for Donor, V for vaccine. Negative control (Lane 13) is PEG with H₂0 and 40 ul of vaccine and negative control (Lane 14) is PEG with PBS.

Fig 5. Quantification of different protein classes and identification of immunoglobulins.

V indicates samples to which influenza vaccine was added. HMW1 refers to the highest molecular weight peak and HMW2 refers to the second peak. Febrile HMW1 eluant, wash-through and Febrile HMW2 eluant and wash-through are not spiked with vaccine. (A) Relative quantities of different classes of protein as calculated through a modified iBAQ-based approach. The total height of each bar is the proportion of MS1 intensity belonging to each identified feature. Samples come from two donors (Donor 1 and Donor 2). (B) Proportion of immunoglobulin mass by class as estimated using a modified iBAQ-based approach and extrapolated from constant peptides only. HA: IgA heavy chain, HD: IgD heavy chain, HE: IgE heavy chain, HG: IgG heavy chain and HM: IgM heavy chain.

Fig 6. Proportion of estimated IgG mass by subclass (excluding contributions by non-specific peptides).

PEG precipitated samples come from two donors (D1 and D2) and V indicates samples to which influenza vaccine was added. HMW1 refers to the highest molecular weight peak and HMW2 refers to the second peak. Febrile HMW1 eluant, wash-through and Febrile HMW2 eluant and wash-through are not spiked with vaccine. HG1: heavy chain of IgG subclass 1, HG2: heavy chain of IgG subclass 2, HG3: heavy chain of IgG subclass 3 and HG4: heavy chain of IgG subclass 4.

Fig 7

Number of MS2 spectra assigned to influenza peptides in each sample. MS1 features matched by run are excluded. Samples come from two donors (D1 and D2). V indicates samples to which influenza vaccine was added. SEC-AP V HMW1 eluant, wash-through, and SEC-AP HMW2 eluant and wash-through are the control samples spiked with vaccine. SEC-AP Febrile HMW1 eluant, wash-through and SEC-AP Febrile HMW2 eluant and wash-through are not spiked with vaccine.