Fractionation of the human plasma proteome for monoclonal antibody proteomics‐based biomarker discovery 2: Antigen identification by dot‐blot array screening

Abstract

Immunization with complex mixtures, like the human plasma resulted in the generation of cloned mAb libraries (PlasmaScan™ and QuantiPlasma™ libraries, with >1000 individual mAbs) reacting with a nonredundant set of antigenic epitopes. mAb proteomics refers to quasi-hypothesis-free profiling of plasma samples with nascent or cloned mAb libraries for the discovery of disease-specific biomarkers. Once mAbs with biomarker potential have been identified, the next task is the determination of cognate antigens recognized by the respective mAbs. To determine the cognate protein antigen corresponding to each individual mAbs in the cloned mAb libraries, we have separated human plasma by consecutive steps of desalting and various chromatography procedures. The process resulted in 783 fractions, which we termed "Analyte Library" (AL). The AL represents the human plasma proteome in relatively low-protein complexity fractions. Here, to determine the utility of the AL, we selected ten plasma proteins and checked for their presence in the fractions. Among the ten cases, the distribution of four selected plasma proteins matched expectations, as these proteins were present only in a few fractions corresponding to their physical, chemical, and biochemical properties. However, in six cases, we observed "smear" -like distribution or complete absence of the proteins, suggesting that protein-protein interactions or protein variants may alter the observed plasma distribution profiles. Nevertheless, we conclude that the AL is an efficient, high throughput tool to complement the mAb biomarker discovery process with cognate protein antigen identification for each mAbs.

Medicinal Chemistry Meets Proteomics: Fractionation of the Human Plasma Proteome

“…The scheme of the Library generation protocol is delineated in Figure 3. The Library fractions proved to be applicable for mAb proteomics-based antigen identification [75]. Another large scale fractionation process employed strong cation exchange chromatography as the first dimension followed by reversed-phase HPLC as second dimension to pre-fractionate the human plasma proteome prior to linear ion trap MS analysis [76].…”