Nitrilotriacetic Acid-Coated Magnetic Nanoparticles as Affinity Probes for Enrichment of Histidine-Tagged Proteins and Phosphorylated Peptides

Abstract

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.

Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles

“…by ligand exchange. Silica coating, however, seems to yield very robust nanoparticles functionalized with primary amines (Buining et al 1997;Li et al 2007). …”

“…In the following, larger Au nanoparticles (Brinas et al 2008) and quantum dots (Gupta et al 2008;Kim et al 2008b) have also been modified with NTA-containing ligands, as well as silanized iron oxide nanoparticles (Li et al 2007) by conjugation of amine-containing NTA to carboxylic groups on the particle surface. After loading the NTA groups with bivalent cations, the modified particles could be used for conjugation to proteins with polyhistidine residues.…”

Microwave Assisted Reactions in Organic Chemistry: A Review of Recent Advances

“…If we load digests with magnetic elements to enhance the microwave absorptivity of the irradiated solutions, similar improvements in terms of digest efficiency/sequence coverage could be observed (Y. Lo et al, 2007;Li et al, 2007).…”

Facile preparation of monolithic immobilized metal affinity chromatography capillary columns for selective enrichment of phosphopeptides

“…Immobilized metal affinity chromatography (IMAC) is one of the widely applied strategies for phosphopeptide 41 ), is used to prepare IMAC materials by which phosphopeptides can be selectively captured due to the high affinity of metal ions for phosphate groups [10][11][12][13][14]. So far, several kinds of matrices, including polymer beads [15,16], the inner wall of capillaries [17,18], and monoliths [19][20][21] have been employed for metal ion immobilization, among which monoliths have exhibited striking features, such as the facility of preparation and modification, adjustable porosity, low-column backpressure [22][23][24], high loading capacity, and fast mass transfer for phosphopeptides, due to the unique porous structure.…”