Characterization of Protein-Hapten Conjugates. 1. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Immuno BSA-Hapten Conjugates and Comparison with Other Characterization Methods

Abstract

Several different low molecular weight haptens were conjugated to BSA to produce immunogens useful for antibody development. The extent of BSA modification due to covalent attachment of hapten was estimated by matrix-assisted laser desorption ionization mass spectrometry. The average number of hapten incorporated to immunogen was determined from the difference in the measured molecular weights of the conjugate from nonmodified BSA. The results from mass spectrometry were compared with results obtained from other more traditional methods of immunogen characterization (UV analysis, trinitrobenzenesulfonic acid titrations, and gel electrophoresis). In each case we were able to calculate the average number of hapten covalently bound to BSA for each synthetically prepared immunogen using matrix-assisted laser desorption ionization mass spectrometry. The other methods presented limitations in certain cases.

2-Morpholinoethylisocyanide as a coupling agent for hapten-protein conjugates

“…The analysis of the homoserine lactone conjugate of BSA indicated that about half of the total amino groups had become unavailable to react with TNBS without apparent incorporation of the hapten. Similar effects have been noted with carbodiimide coupling of some haptens (Adamczyk et al 1994), and could be due to changes in tertiary structure, aggregation, crosslinking of the carrier or the formation of non-covalent adducts, N-acyl urea adducts (for carbodiimides) or uncharacterized side reactions. The hapten density of protein conjugates is known to affect their immunogenic properties, with relatively low densities favoring a high affinity, predominantly IgG response (Klaus & Cross 1974), owing to the relative strength of interactions with T-and B-cell antigen receptors.…”

Characterization of Protein−Hapten Conjugates. 2. Electrospray Mass Spectrometry of Bovine Serum Albumin−Hapten Conjugates

“…TNBS titration, UV-difference spectroscopy, gel electrophoresis). Each of these methods have their own limitations (17). Classical biochemical methodologies, however, can be greatly augmented or replaced by bioanalytical mass spectrometry.…”

“…Now, mass spectrometry techniques (FAB, MALDI, ESI) have emerged as powerful tools for directly observing the mass of modified proteins (13,14) and thereby offer a means of quantifying the extent and type of modification (15,16). Recently, we compared MALDI-MS with other more traditional methods for the characterization of a series of conjugates (17). At that time, MALDI-MS appeared to be the method of choice for the direct observation of the average molecular weight of the derivatized protein.…”

Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin–fluorescein isothiocyanate conjugates

“…According to the manufacturer's specifications, the BSA-FITC conjugate has a degree of substitution of 7-12 moles of FITC per mole albumin measured using absorbance spectroscopy. This kind of discrepancies between methods has been observed earlier [17] and probably depends on that MALDI only detects the covalent conjugates in contrast to other methods that may additionally detect non-covalent complexes. On the other hand, the establishment of the centroid peak in the MALDI analysis is obstructed by the broad peaks obtained, which influence the precision of the ratio determination.…”

“…It is also well studied and used as a model protein for a diversity of purposes including protein-hapten conjugate studies [17,18]. Consequently, FITC-bound serum albumin is a commonly used fluorescent marker and can be bought already labeled.…”