Hydrogen exchange/electrospray ionization mass spectrometry studies of structural features of proteins and protein/protein interactions.

The rate at which amide hydrogens located at the peptide backbone in protein/protein complexes undergo hydrogen/deuterium exchange is highly dependent on whether the amide groups participate in binding. Here, a new mass spectrometric method is presented in which this effect is utilized for the characterization of protein/ligand binding sites. The information obtained is which region within the protein participates in binding. The method includes hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange. After this procedure those backbone amide groups that participate in protein binding are protected from back exchange and therefore still deuterated. These regions were then identified by peptic proteolysis, fast microbore high-performance liquid chromatography separation, and electrospray ionization mass spectrometry. The approach has been applied to the investigation of structural features of insulin-like growth factor I (IGF-I) and the interaction of insulin-like growth factor I with IGF-I binding protein 1. The data show that the approach can provide information on the location of the hydrophobic core of IGF-1 and on two regions that are mainly involved in binding to IGF-I binding protein 1. The data are consistent with results obtained with other approaches. The amount of sample required for one experiment is in the subnanomolar range.

Matrix-assisted laser desorption/ionization mass spectrometry of proteins extracted directly from sodium dodecyl sulphate-polyacrylamide gels.

A new strategy for the characterization of Coomassie Brilliant Blue stained SDS-PAGE separated proteins by UV-MALDI-MS is reported. The proteins are extracted directly from the polyacrylamide gel by treatment with an organic solvent mixture consisting of formic acid, acetonitrile, isopropanol and water in an ultrasonic bath. A fraction of the supernatant is then mixed directly with the matrix solution and measured by MALDI-MS. High quality spectra could be obtained from gels which were loaded with 6 pmol of myoglobin. Compared to other methods based on electroblotting or electroelution this method is much simpler and less time consuming. The sensitivity is higher than or comparable to the Coomassie Blue staining procedure for proteins up to about 25 kDa. Another advantage is that mass shifts due to charging effects of the membranes, which are common if membranes are mounted directly on the sample target, can be avoided. However, all proteins studied showed slightly higher masses than expected which reduces mass accuracy to 0.2-0.3%. This is presumably partly due to formylation of serine or threonine residues during incubation in formic acid. Gel electrophoresis induced modifications can contribute as well. The possibility of further characterizing the remaining part of the supernatant after extraction by means of proteolytic digestion is also demonstrated. The knowledge of both molecular weight of the whole protein and of the proteolytic fragments increases specificity for protein identification by searching in sequence databases.

Isolation and biochemical characterization of highly purified Escherichia coli molecular chaperone Cpn60 (GroEL) by affinity chromatography and urea-induced monomerization.

Isolated Escherichia coli molecular chaperone Cpn60 (GroEL) has been further purified from tightly bound substrate polypeptides by two different procedures: (i) group-specific affinity chromatography by using the triazine dye Procion yellow HE-3G as affinity ligand, and (ii) urea-induced monomerization and subsequent chromatography. Procion yellow binds specifically to aromatic amino-acid side chains present in the majority of proteins, but has no affinity to GroEL because of its low content of aromatic residues. Some GroEL-bound polypeptides are buried within the aqueous cavity of the GroEL oligomer, whereas others are exposed on its surface and available for affinity-ligand interactions and the complex is thereby retarded on Procion yellow columns. Pure substrate-free GroEL was obtained after ion-exchange chromatography of GroEL monomers followed by reassembly of the purified monomers into functional GroEL oligomers. The final preparation contained no substrate polypeptides bound to GroEL as judged by electrophoretic analysis and lack of tryptophan fluorescence. GroEL preparations also displayed two equally strong bands on native electrophoresis suggesting the presence of two conformers. Monomers of GroEL showed heterogeneity with respect to isoelectric point and molecular mass when analysed by MALDI-MS and electrophoresis under native and denaturing conditions respectively. By use of MALDI-MS, highly accurate molecular masses of wild-type and a truncated form of GroEL were determined and verified, by comparison with their respective gene sequences.