Facets of individual-specific health signatures determined from longitudinal plasma proteome profiling

Background: Precision medicine approaches aim to tackle diseases on an individual level through molecular profiling. Despite the growing knowledge about diseases and the reported diversity of molecular phenotypes, the descriptions of human health on an individual level have been far less elaborate. Methods: To provide insights into the longitudinal protein signatures of well-being, we profiled blood plasma collected over one year from 101 clinically healthy individuals using multiplexed antibody assays. After applying an antibody validation scheme, we utilized > 700 protein profiles for in-depth analyses of the individuals’ short-term health trajectories. <p<Findings: We found signatures of circulating proteomes to be highly individual-specific. Considering technical and longitudinal variability, we observed that 49% of the protein profiles were stable over one year. We also identified eight networks of proteins in which 11 242 proteins covaried over time. For each participant, there were unique protein profiles of which some could be explained by associations to genetic variants. Interpretation: This observational and non-interventional study identifyed noticeable diversity among clinically healthy subjects, and facets of individual-specific signatures emerged by monitoring the variability of the circulating proteomes over time. To enable more personal hence precise assessments of health states, longitudinal profiling of circulating proteomes can provide a valuable component for precision medicine approaches

Characterization of antibody specificity using peptide array technologies

Antibodies play an important role in the natural immune response to invading pathogens. The strong and specific binding to their antigens also make them indispensable tools for research, diagnostics and therapy. This thesis describes the development of methods for characterization of an- tibody specificity and the use of these methods to investigate the polyclonal antibody response after immunization. Paper I describes the development of an epitope-specific serum fractionation technique based on epitope map- ping using overlapping peptides followed by chromatographic separation of polyclonal serum. This technique together with another epitope mapping technique based on bacterial display of protein fragments were then used to generate antibody sandwich pairs (Paper I), investigate epitope variations of repeated immunizations (Paper II) and to determine the ratio of antibodies targeting linear and conformational epitopes of polyclonal antibodies (Paper III). Paper IV describes the optimization of in situ-synthesized high-density peptide arrays for epitope mapping and how different peptide lengths influ- ence epitope detection and resolution. In Paper V we show the development of planar peptide arrays covering the entire human proteome and how these arrays can be used for epitope mapping and off-target binding analysis. In Paper VI we show how polyclonal antibodies targeting linear epitopes can be used for peptide enrichment in a rapid, absolute protein quantification protocol based on mass spectrometry. Altogether these investigations demonstrate the usefulness of peptide arrays for fast and straightforward characterization of antibody specificity. The work also contributes to a deeper understanding of the polyclonal anti- body response obtained after immunization with recombinant protein frag- ments.QC 20141111</p

High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays

Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level

Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes

<div><p>A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.</p> </div

Dissecting antibodies with regards to linear and conformational epitopes.

An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets

Schematic overview of the epitope mapping methods used in the study.

<p>(<b>A</b>) Epitope mapping using bacterial display, in which the target gene is fragmented and a library of clones is expressed on <i>S. carnosus.</i> The cell displayed peptide library is assayed for binding to the antibody using a flow cytometer Binding clones are sorted, sequenced and aligned back to the antigen sequence in order to conclude epitopes. (<b>B</b>) The sequencing of the flow-sorted libraries is used to determine the epitope regions. To the left, a typical FACS dot plot showing sorting of a cell displayed library incubated with the investigated antibody. The colored bars to the right show aligned binding sequences from the different sorted populations and on top the consensus epitopes derived from the alignment. (<b>C</b>) Epitope mapping using peptide bead arrays with overlapping peptides, spanning the antigen sequence, coupled to color coded beads. Antibody binding towards the peptides is evaluated using a flow cytometer instrument and epitope regions are identified on the antigen. (<b>D</b>) A schematic view of how binding profiles are used to determine epitope regions.</p

In-depth analysis of polyclonal antibodies towards TYMP.

<p>The three polyclonal antibodies from the separate immunizations of a recombinant fragment of TYMP were mapped to reveal the major epitopes, which are highlighted in different colors (Bead array). The peptides corresponding to the major epitopes were synthesized and used for affinity purification. The amount of antibodies eluted from the peptide-specific affinity capture was determined and illustrated in colors corresponding to each peptide specific antibody (Affinity capture). The “Relative bead array activity” was calculated as the mean fluorescent intensity (MFI) detected for each peptide-covered bead per µg epitope-specific antibody in the purified polyclonal antiserum. The affinity purified antibody fractions were diluted to the same concentration and subsequently used in a Western blot assay and the intensity of the correct sized band was determined using software ImageJ (Relative WB activity).</p

Schematic overview of the epitopes determined by the two methods and epitope prediction for the corresponding antigen.

<p>(i) Plots show the hydrophilicity value predicted by Hopp and Woods (blue) and antibody response value by Rockberg and Uhlen (orange) for the six antigens analyzed by the both epitope mapping methods. The prediction plots are illustrated above the consensus epitope sequence from the suspension bead array shown in panel (ii) and the bacterial display in panel (iii) from the different antibodies shown as colored bars (Ab1: red, Ab2: blue and Ab3: green). The x-axis indicates the amino acid position in antigen sequence and the y-axis indicates the hydrophilicity score.</p