Mass spectrometric mapping of fibrinogen conformations at poly(ethylene terephthalate) interfaces☆

Abstract

We have characterized the adsorption of bovine fibrinogen onto the biomedical polymer polyethylene terephthalate (PET) by performing mass spectrometric mapping with a lysine-reactive biotin label. After digestion with trypsin, MALDI-TOF mass spectrometry was used to detect peptides from biotinylated bovine fibrinogen, with the goal of identifying lysines that were more accessible for reaction with the chemical label after adsorption. Peptides within domains that are believed to contribute to heparin binding, leukocyte activation, and platelet adhesion were found to be biotin labeled only after bovine fibrinogen adsorbed to the PET surface. Additionally, the accessibility of lysine residues throughout the entire molecule was observed to increase as the concentration of the adsorbing bovine fibrinogen solution decreased, suggesting that the proximity of biologically active motifs to hydrophilic residues leads to their exposure. The surface area per adsorbed bovine fibrinogen molecule was quantified on PET using optical waveguide lightmode spectroscopy (OWLS), which revealed higher surface densities for bovine fibrinogen adsorbed from higher concentration solutions. By measuring changes in both the identity and conformation of proteins that adsorb from complex mixtures such as blood or plasma, this technique may have applications in fundamental studies of protein adsorption and may allow for more accurate predictions of the biocompatibility of materials.

Experimental characterization of adsorbed protein orientation, conformation, and bioactivity

“…51 Although, the applicability of this technique on adsorbed protein has been previously investigated by many groups, its use has been restricted to determining the labeling profile of just one type of amino acid because of problems related to differences in MS intensities that result from each of the different labeling processes. [27][28][29][30] However, the labeling profile for a single amino acid type provides very limited information for the assessment of the orientation and conformational changes of an adsorbed protein. Therefore, to provide more complete coverage for a given protein, our group recently developed methodology to combine labeling results from multiple target amino acid types applied to a single protein.…”

“…15,17 Some of the methods that can provide information on adsorbed protein orientation and tertiary (and quaternary) structure include fluorescence, [18][19][20][21] time-of-flight secondary-ion mass spectrometry, [22][23][24] nuclear magnetic resonance spectroscopy (NMR), 25,26 and amino acid labeling/mass spectrometry (AAL/MS). [27][28][29][30][31] Methods for the determination of secondary structure of adsorbed proteins include Fourier transform infrared spectroscopy, 32,33 surface enhanced Raman scattering, 34,35 and circular dichroism spectropolarimetry (CD). 27,[36][37][38][39] Unfortunately, as the size of the protein increases, many of the spectral signatures that are needed for tertiary structure determination using fluorescence and NMR overlap, introducing much subjectivity into the analyses, thus making it difficult to accurately interpret the configuration of the adsorbed protein.…”

“…In contrast, MS has shown great promise in characterizing the adsorbed configuration of both large and small proteins at a molecular level, especially when used with AAL. [27][28][29][30][31] After evaluating several of these types of methods, our group has specifically focused on the development and adaptation of CD and AAL/MS for the characterization of adsorbed protein orientation and structure, and we will therefore focus on these methods for this paper. Readers are encouraged to refer to the above cited references for the application of the other types of methods that are noted above.…”

Probing the Conformation and Orientation of Adsorbed Enzymes Using Side-Chain Modification

“…The selective modification of amino acids has seen widespread use for more than 40 years in the study of protein structure and function because of its simplicity and effectiveness 26 and has recently been applied to probe the effect of adsorption on the structure of fibrinogen by Scott and Elbert by the modification of lysine residues. 27 Tryptophan (Trp) is an ideal residue to label for our purposes since solvent accessible Trps are located near the active site of both HEWL and GOx as well as within their normally solvent-inaccessible hydrophobic core. Solvent-accessible Trps can be covalently modified under mild conditions and physiological pH using dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS; 294.2 g/mol) and the extent of modification can be determined spectrophotometrically in basic solutions (pH ≥ 10).…”

Biocompatibility and Biological Performance of the Improved Polyurethane Membranes for Medical Applications

“…There are many data concerning mechanisms of protein adsorption on different surfaces (Gray, 2004;Scott & Elbert, 2007;Van Tassel, 2006;Wilson et. al., 2005).…”