Formaldehyde is frequently used to inactivate, stabilize, or immobilize proteins. The treatment results in a large variety of chemical modifications in proteins, such as the formation of methylol groups, Schiff bases, and methylene bridges. The purpose of the present study was to identify the stable formaldehyde-induced modifications in a small protein. Therefore, insulin was treated with excess formaldehyde (CH2O) or deuterated formaldehyde (CD2O). In a separate experiment, insulin was modified by formaldehyde (CH2O vs CD2O) and glycine. The mixture of CH2O-treated and CD2O-treated insulin was digested by the proteinase Glu-C. The peptide fragments obtained were analyzed by liquid chromatography-mass spectrometry (LC-MS). Seven intramolecular cross-links were identified in formaldehyde-treated insulin. Furthermore, eight out of the sixteen potentially reactive sites of the insulin molecule were modified by incubation with formaldehyde and glycine. Both the location and the chemical nature of the modifications could be assigned based on the mass increase of potential adducts as elucidated in our previous study (B. Metz et al. (2004) J. Biol. Chem. 279, 6235-6243). To confirm the assigned structures, LC-MS measurements with collision-induced dissociation (LC-MS/MS) were performed on insulin fragments. The results of the LC-MS/MS analyses agreed excellently with the assignments. The study showed that arginine, tyrosine, and lysine residues were very reactive. However, eight theoretically reactive residues did not show detectable modifications, probably because of their low intrinsic reactivity, inaccessibility, or both. The asparagine, glutamine, and histidine residues were not converted in insulin. The N-termini of insulin were partly converted to the expected imidazolidinone adducts, indicating that the protein conformation affects the accessibility and reactivity of these residues. In conclusion, this study shows that, based on our current insights in the chemistry of the reactions between proteins and formaldehyde, we are able to elucidate the location and nature of formaldehyde-induced modifications in a small protein. The approach followed in this study may be generally applicable to larger formaldehyde-treated proteins, such as toxoids used in vaccines.

The use of mammalian cells for therapeutic applications is finding its way into modern medicine. However, modification or “training” of cells to make them suitable for a specific application remains complex. By envisioning a chemical toolbox that enables specific, but straight-forward and generic cellular functionalization, we investigated how membrane-receptor (pre)targeting could be combined with supramolecular host-guest interactions based on β-cyclodextrin (CD) and adamantane (Ad). The feasibility of this approach was studied in cells with membranous overexpression of the chemokine receptor 4 (CXCR4). By combining specific targeting of CXCR4, using an adamantane (Ad)-functionalized Ac-TZ14011 peptide (guest; KD = 56 nM), with multivalent host molecules that entailed fluorescent β-CD-Poly(isobutylene-alt-maleic-anhydride)-polymers with different fluorescent colors and number of functionalities, host-guest cell-surface modifications could be studied in detail. A second set of Ad-functionalized entities enabled introduction of additional surface functionalities. In addition, the attraction between CD and Ad could be used to drive cell-cell interactions. Combined we have shown that supramolecular interactions, that are based on specific targeting of an overexpressed membrane-receptor, allow specific and stable, yet reversible, surface functionalization of viable cells and how this approach can be used to influence the interaction between cells and their surroundings.

We present a versatile strategy for constructing particle-in-a-box-in-a-box systems by assembling dendrimer-encapsulated gold nanoparticles into dendrimicelles.