Cleavable ester linked magnetic nanoparticles for labeling of solvent exposed primary amine groups of peptides/proteins

Covalent labeling of solvent exposed amino acid residues using chemical reagents/crosslinkers followed by mass spectrometric analysis can be used to determine the solvent accessible amino acids of a protein. A variety of chemical reagents containing cleavable bonds were developed to label abundantly found lysine residues on the surface of protein. To achieve efficient separation of labeled peptides prior to mass spectrometric analysis, magnetic nanoparticles can be decorated with amino acid reactive functional groups and utilized for quick recovery of labeled peptides. [1] In this work, iron oxide magnetic nanoparticles (Fe3O4 MNPs) were synthesized by thermal decomposition method and coated with silica (SiO2@Fe3O4 MNPs) by reverse micro emulsion approach. The Fe3O4 MNPs and SiO2@Fe3O4 MNPs were characterized by TEM and XRD. The SiO2@Fe3O4 MNPs were further coated with amine groups and conjugated to N-hydroxysuccinimidyl (NHS) ester groups via a cleavable ester bond. Fluorescence based qualitative analysis of ester linked NHS ester modified SiO2@Fe3O4 MNPs was performed to confirm the presence of NHS ester group. The active NHS ester sites on the surface of SiO2@Fe3O4 MNPs were determined by depletion approach and found to be 694 active sites per 1 mg of SiO2@Fe3O4 MNPs. Free amine groups of a small peptide, ACTH (4–11) were labeled by ester linked, NHS ester modified SiO2@Fe3O4 MNPs under physiological conditions. Superparamagnetic nature of SiO2@Fe3O4 MNPs allowed quick and efficient magnetic separation of labeled peptides from the solution. The ester bond was further cleaved to separate labeled peptides followed by mass spectrometric analysis. The ester linked, NHS ester modified SiO2@Fe3O4 MNPs introduced a mass shift of 115.09 Da on amine groups of ACTH (4–11), which was confirmed by mass spectrometry.

Superparamagnetic nature of SiO 2 @Fe 3 O 4 MNPs allowed quick and efficient magnetic separation of labeled peptides from the solution. The ester bond was further cleaved to separate labeled peptides followed by mass spectrometric analysis. The ester linked, NHS ester modified SiO 2 @Fe 3 O 4 MNPs introduced a mass shift of 115.09 Da on amine groups of ACTH (4)(5)(6)(7)(8)(9)(10)(11), which was confirmed by mass spectrometry.
& 2015 Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). MNPs. Solvent exposed free amine residues of peptides were labeled using cleavable ester linked NHS ester linked silica coated iron oxide magnetic nanoparticles. The labeling reaction was performed under physiological conditions to preserve the native structure of proteins. The ester bond was subsequently cleaved followed by magnetic separation of nanoparticles Experimental features

Specifications
The label generated on the solvent exposed free amine groups of peptides and proteins were identified by mass spectrometric analysis.

Data, experimental design and methods
The data shown here is divided into four major steps: a) synthesis and characterization of SiO 2 @Fe 3 O 4 MNPs, b) synthesis of ester linked NHS ester modified SiO 2 @Fe 3 O 4 MNPs, c) fluorometric quantification of active NHS ester sites on the surface of SiO 2 @Fe 3 O 4 MNPs and, d) labeling and identification of primary amine groups of ACTH (4-11) using ester linked NHS ester modified SiO 2 @Fe 3 O 4 MNPs.

Synthesis of iron oxide nanoparticles
Iron oxide nanoparticles were synthesized as described [2] and characterized by XRD (Fig. 1).

Silica coating of Fe 3 O 4 MNPs by reverse micro-emulsion approach (scheme 1)
Silica coating was performed as reported earlier. [3] The Fe 3 O 4 MNPs (400 mL of 10 mg/mL) were dissolved in cyclohexane (4 mL) and Igepal-CO-520 (0.247 g) then sonicated for 15 min. The suspension was further mixed with tetraethyorthosilicate (25 mL) and sonicated again for 10 min. In the last step, ammonium hydroxide (50 mL) was added and sonicated for 15 min. The suspension was stirred using a magnetic stirrer at room temperature for 24 h. The SiO 2 @Fe 3 O 4 MNPs were magnetically recovered, washed using ethanol several times, and dried at room temperature. Amine groups were introduced on the surface of SiO 2 @Fe 3 O 4 MNPs using amine containing silane coupling reagent [4]. The SiO 2 @Fe 3 O 4 MNPs (10 mg) were resuspended in ethanol (10 mL) followed by 20 min of sonication. In the next step, APTES (95%, 100 μL) was added dropwise and the mixture was mechanically stirred at room temperature for 24 h. Amine modified Fe 3 O 4 SiO 2 @Fe 3 O 4 MNPs were magnetically separated, washed several times with ethanol, and air dried at room temperature.

Synthesis of cleavable linked, NHS ester modified SiO 2 @Fe 3 O 4 MNPs (scheme 1)
Amine modified SiO 2 @Fe 3 O 4 MNPs (1 mg) were mixed with ethanol (100 μL), followed by sonication for 10 min. EGS (12 mg in μL DMSO, 100 final conc. 0.13 M) was added dropwise to the solution of amine modified SiO 2 @Fe 3 O 4 MNPs and allowed to react for 20 min at room temp. EGS modified SiO 2 @Fe 3 O 4 MNPs were recovered by magnetic separation, washed with ethanol, and dried under vacuum.
Presence of NHS ester was determined by conjugating dansylcadaverine and mesuring the fluoscence of dansylcadaverine conjugated SiO 2 @Fe 3 O 4 MNPs.

Labeling amine groups of peptides/proteins using cleavable ester linked, NHS ester modified SiO 2 @Fe 3 O 4 MNPs
Labeling of ACTH (4-11), BSA and β-lactoglobulin was performed by following a protocol as reported earlier [5] with minor modifications. Protein sample (BSA or β-lactoglobulin, 10 μL, 10 mg/mL) solution was mixed with ester cleavable, NHS ester modified SiO 2 @Fe 3 O 4 MNPs followed by addition of PBS, pH ¼7.4 (190 μL). The mixture was allowed to stir at room temperature for 40 min. The mass spectra of a doubly charged, both amine modified ACTH (4-11) is shown in Fig. 3. The b and y ion were manually calculated and matched with major peaks in the spectra. 5.6. Mass spectrometric analysis of labeled ACTH (4-11), BSA and β-lactoglobulin Chromatographic separation was performed by using a chip consisting of a 160 nL enrichment column and a 150 mm analytical column packed with C18, 5 m beads with 300 Å pores. The sample (2 μL) was transferred to the enrichment column via the capillary pump. Capillary pump was operated     4. Fluorescence spectra of danylcadaverine before conjugation to cleavable ester linked NHS ester modified SiO 2 @Fe 3 O 4 MNPs (blue) and danylcadaverine after conjugation to cleavable ester linked NHS ester modified SiO 2 @Fe 3 O 4 MNPs (red). measurements (Fig. 4). The quantity of conjugated dansylcadaverine was determined by subtracting the quantity of remaining dansylcadaverine after conjugation to NHS ester modified SiO 2 @Fe 3 O 4 MNPs from the initial quantity of dansylcadaverine.