Magnetic nanoparticle-based platform for characterization of histidine-rich proteins and peptides.

In this study, we developed a platform that can be used to rapidly enrich polyhistidine(His)-tagged proteins/peptides from complex samples selectively using the Fe3O4@Al2O3 magnetic nanoparticles (MNPs) as the affinity probes. At pH 7, the dissociation constant between poly-His, i.e., His6, and the Fe3O4@Al2O3 MNPs was ~10(-5) M and the trapping capacity was ~100 nmol/mg for His6. Enrichment was achieved by vigorously mixing the sample solution (<2 μL) and the MNPs (1-3 μg) by pipetting directly onto a matrix-assisted laser desorption/ionization (MALDI) plate for 10 s. The time for the enrichment and the sample volume required for analysis are therefore greatly reduced. After enrichment, the MNP-target species conjugates were promptly isolated by positioning a magnet on the edge of the sample well to aggregate the conjugates into a small spot within ~5 s so that the nontarget species could be easily removed. Additionally, the problem of finding "sweet spots" on the target species during the MALDI mass spectrometry (MS) analysis was greatly reduced by magnetically isolating the target species on the MALDI plate. The limit of detection for His6 was, therefore, as low as ~400 amol. His6 and AHHAHHAAD AHHAHHAAD spiked in a protein digest and in human plasma, respectively, were used as the samples to demonstrate the practicability of this approach in selective enrichment of His-rich peptides from complex samples. We also characterized His6-tagged proteins enriched on-plate by the Fe3O4@Al2O3 MNPs followed by on-plate tryptic digestion, selective enrichment, and MALDI-MS analysis. This approach can be used to determine quickly whether His6-tagged species are present in a sample. In addition, cell lysates containing recombinant Shiga-like toxins tagged with His6 were used as the samples to further demonstrate that the feasibility of this approach in analyzing very complex samples. The entire analysis process, including the on-plate enrichment and enzymatic digestion followed by MALDI-MS analysis, can be completed within 10 min.