Nitrilotriacetic acid-coated magnetic nanoparticles as affinity probes for enrichment of histidine-tagged proteins and phosphorylated peptides.
نویسندگان
چکیده
We herein demonstrate superparamagnetic Fe3O4 nanoparticles coated with nitrilotriacetic acid derivative (NTA) that can bind with different immobilized metal ions are capable of probing diverse target species. Immobilized Ni(II) on the surfaces of the NTA-magnetic nanoparticles have the capability of selectively trapping histidine (His)-tagged proteins such as a mutated streptopain tagged with 6x His, i.e., C192S (MW approximately 42 kDa), from cell lysates. Enrichment was achieved by vigorously mixing the sample solution and the nanoparticles by pipetting in and out of a sample vial for only 30 s. After enrichment, the probe-target species could be readily isolated by magnetic separation. We also characterized the proteins enriched on the affinity probes using on-probe tryptic digestion under microwave irradiation for only 2 min, followed by matrix-assisted laser desorption/ionization mass spectrometry analysis. Using this enrichment and tryptic digestion, the target species can be rapidly enriched and characterized, reducing the time required for carrying out the complete analysis to less than 10 min. Furthermore, when either Zr(IV) or Ga (III) ions are immobilized on the surfaces of the NTA-magnetic nanoparticles, the nanoparticles have the capability of selectively enriching phosphorylated peptides from tryptic digests of alpha-, beta-caseins, and diluted milk. The detection limit for the tryptic digests of alpha- and beta-caseins is approximately 50 fmol.
منابع مشابه
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 wa...
متن کاملAffinity surface-assisted laser desorption/ionization mass spectrometry for peptide enrichment.
In this paper, we report on the functionalization of silicon nanostructured (NanoSi) surface with an organic layer of nitrilotriacetic acid (NTA) and its subsequent use as an affinity surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) interface for histidine-tagged peptide enrichment and mass spectrometry analysis. The NTA terminal groups are immobilized onto the NanoSi s...
متن کاملFluorescent receptor-immobilized silica-coated magnetic nanoparticles as a general binding agent for histidine-tagged proteins.
The histidine-tagged protein binding capacity and purification efficiencies of TSMNPs (terpyridine receptor-immobilized silica-coated magnetic nanoparticles) is described.
متن کاملRapid enrichment of phosphopeptides and phosphoproteins from complex samples using magnetic particles coated with alumina as the concentrating probes for MALDI MS analysis.
In this study, we used nanocomposite magnetic particles coated with alumina as the affinity probes to selectively concentrate phosphorylated peptides and proteins from a low volume of sample solution. Tryptic digest products of phosphoproteins including alpha and beta-caseins, human protein phosphatase inhibitor 1, nonfat milk, egg white, and a cell lysate were used as the samples to demonstrat...
متن کاملSpecific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding
Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages' pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Analytical chemistry
دوره 79 19 شماره
صفحات -
تاریخ انتشار 2007