Bovine serum albumin coverage

J.E. Bruce, G.A. Anderson, J. Wen, R. Harkewicz, R.D. Smith, High-mass-measurement accuracy and 100% sequence coverage of enzymatically digested bovine serum albumin from an ESI-FTICR mass spectrum. Anal. Chem., 71 (1999) 2595. 

Using this method, we constructed a number of dynamic adsorption isotherms for several proteins on different surfaces. Here we present information for two proteins. The adsorption isotherm for bovine serum albumin (BSA) (87% monomer, 13% dimer described by the supplier as 100% monomer) is shown in Figure 6. A plateau concentration for BSA on an amine/ silane surface was 3.5 mg/m2, which is comparable with monolayer adsorption reported previously in a static system (3). [The effect of flow rate, ionic strength, and temperature is reported elsewhere (18).] Perhaps more interesting are the data for plasma fibronectin (produced and purified extensively in our laboratory), because adsorption characteristics of this protein have not been reported elsewhere. Since it is a protein intimately involved in cellular adhesion, its surface behavior is particularly relevant to implant biocompatibility. We found a plateau uptake of approximately 7.0 mg/m2 on both amine and dimethyldimethoxymethylsilane, and based on the assumption of monolayer coverage and a published axial ratio (20), we calculated that the molecule has dimensions of approximately 130 X 80 X 100 A. 

The performance of the device was demonstrated with the separation of a tryptic digest of bovine serum albumin (BS A) in the reversed-phase mode using a gradient of acetonitrile in aqueous solution of trifiuoroacetic acid as the mobile phase. The separated peptides were detected by mass spectrometer. Figure 47.6 shows both the device and separation of a sample containing 5 pmol of peptides. Comparison with a database revealed an excellent amino acid sequence coverage of 70%. 

Raje and Pinto [31] measured the heat of adsorption and its dependence on surface coverage for protein ion-exchange systems of bovine serum albumin and ovalbumin. Experimental data showed that protein adsorption is endothermic for both systems, which suggests that the process is entropicaUy driven. Therefore, it is essential to include the entropic contribution (i.e., hydrophobic interactions) in modeling equihbrium behavior. 

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