Identifying nonspecific ligand binding in electrospray ionization mass spectrometry using the reporter molecule method.

The application of the reporter molecule (M(rep)) method for identifying nonspecific complexes in the ES-MS analysis of protein-ligand and DNA-ligand interactions in vitro is described. To test the reliability of the method, it was applied to the ES-MS analysis of protein-carbohydrate complexes originating from specific interactions in solution and from nonspecific interactions in the ES process. These control experiments confirm the basic assumptions underlying the M(rep) method, namely that nonspecific ligand binding is a random process, and that the ES droplet histories for specific and nonspecific complexes are distinct.

Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile.

The biological and ligand-binding properties of recombinant C-terminal cell-binding domains (CBDs) and subdomains of the two large exotoxins, Toxin A (TcdA) and Toxin B (TcdB) expressed by Clostridium difficile were examined in the hemagglutination and Verocytotoxicity neutralization assays and by qualitative affinity chromatography using Sepharose-linked alpha Gal(1,3)betaGal(1,4)beta Glc as well as the direct electrospray ionization mass spectrometry (ES-MS) assay. These studies revealed that, whereas the full-length TcdA CBD agglutinated rabbit erythrocytes, neutralized TcdA-mediated Vero cell death and bound to alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose, the TcdB CBD was inactive in these functional assays. Moreover, retention by alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose corresponded to the number of available TcdA subdomain ligand-binding sites.

Method for identifying nonspecific protein-protein interactions in nanoelectrospray ionization mass spectrometry.

The nonspecific self-association of proteins in nanoflow electrospray ionization mass spectrometry (nanoES-MS), and the influence of experimental conditions thereon, are investigated using the protein ubiquitin (Ubq) as a model system. Extents of nonspecific protein association generally increase with protein concentration and, interestingly, with decreasing ES spray potential. The extent of self-association is also sensitive to the duration of the accumulation event in an external rf hexapole.

The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin.

Synthetic N-acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc-specific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells.

Method for stabilizing protein-ligand complexes in nanoelectrospray ionization mass spectrometry.

The interaction between the bovine pancrease trypsin (Tryp) and its competitive inhibitor benzamidine (1), in solution and the gas phase, is investigated using nanoflow electrospray ionization (nanoES) and Fourier transform ion cyclotron resonance mass spectrometry. In a recent study (Clark, S.M.

Method for distinguishing specific from nonspecific protein-ligand complexes in nanoelectrospray ionization mass spectrometry.

A new methodology for distinguishing between specific and nonspecific protein-ligand complexes in nanoelectrospray ionization mass spectrometry (nanoES-MS) is described. The method involves the addition of an appropriate reference protein (P(ref)), which does not bind specifically to any of the solution components, to the nanoES solution containing the protein(s) and ligand(s) of interest. The occurrence of nonspecific protein-ligand binding is monitored by the appearance of nonspecific (P(ref) + ligand) complexes in the nanoES mass spectrum.

Blackbody infrared radiative dissociation of nonspecific protein-carbohydrate complexes produced by nanoelectrospray ionization: the nature of the noncovalent interactions.

Gas-phase thermal dissociation experiments, implemented with blackbody infrared radiative dissociation (BIRD) and Fourier transform ion cyclotron resonance mass spectrometry, have been performed on a series of protonated and deprotonated 1:1 and protonated 1:2 protein-carbohydrate complexes formed by nonspecific interactions during the nanoflow electrospray (nanoES) ionization process. Nonspecific interactions between the proteins bovine carbonic anhydrase II (CA), bovine ubiquitin (Ubq), and bovine pancreatic trypsin inhibitor and several carbohydrates, ranging in size from mono- to tetrasaccharides, have been investigated. Over the range of temperatures studied (60-190 degrees C), BIRD of the protonated and deprotonated complexes proceeds exclusively by the loss of the carbohydrate in its neutral form.