ABSTRACT
Mass spectrometry (MS) is one of the most powerful instrumental techniques for protein analysis. The electrospray ionization (ESI) approach is known to be very gentle and at the same time compatible with liquid separation techniques such as HPLC and CE. However, ESI is known to be susceptible to salts and impurities, which often cause a dramatic decrease in sensitivity due to the suppression of the ionization of the product of interest. For this reason, LC-ESI-MS coupling has so far been largely limited to reversed-phase chromatography with its hydro-organic mobile phases. Other chromatographic techniques are typically "linked" to ESI-MS by time consuming, off-line desalting steps. On-line microdialysis has been proposed as a solution to this dilemma. In this paper, we introduce an improved microdialysis system, which enlarges the number of putative applications, thus allowing chromatographic separations of biological compounds to be directly coupled to MS detection with little to no loss in time or chromatographic resolution. Examples include separations by affinity, ion-exchange and size-exclusion chromatography, all of which were connected successfully to the ESI-MS detector via the on-line microdialyzer. We propose that, using this system, any kind of chromatography technique can be coupled to ESI-MS, thus enabling for example application in quality control or process monitoring of many bioproduction and downstream processes.
Subject(s)
Microdialysis/instrumentation , Proteins/chemistry , Animals , Cattle , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Hemoglobins/chemistry , Indicators and Reagents , Molecular Weight , Myoglobin/chemistry , Proteins/isolation & purification , Glycine max/chemistry , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Ultraviolet , Staphylococcal Protein A/chemistry , Staphylococcal Protein A/isolation & purification , Trypsin Inhibitors/chemistryABSTRACT
Proteolytic activation of retroviral envelope glycoprotein precursors occurs at the carboxyl side of a consensus motif consisting of the amino acid sequence (Arg/Lys)-Xaa-(Arg/Lys)-Arg. Synthetic peptides spanning the processing sites of HIV-1/2 and SIV glycoprotein precursors were examined for their ability to be cleaved by the subtilisin-like endoproteases kexin and furin. To determine the potential role of secondary structure on proteolytic activation, we examined the secondary structure of synthetic peptides by circular dichroism and NMR spectroscopy. The results indicate that (i) the peptides were correctly cleaved by kexin and furin and therefore could be used as specific substrates for the purification and characterization of the lymphocyte endoprotease(s) responsible for proteolytic processing of precursors; (ii) the regions surrounding the cleavage sites could be characterized by their flexibility in aqueous solutions. However, a loop has been shown to be a determinant for the specificity of the interaction between the enzyme and its substrate as determined by molecular modeling. Furthermore, we determine and propose a possible structure of the cleavage site which fits to the active site of the modeled furin.
Subject(s)
Gene Products, env/chemistry , HIV Envelope Protein gp160/chemistry , HIV-1 , Proprotein Convertases , Protein Conformation , Protein Precursors/chemistry , Protein Structure, Secondary , Saccharomyces cerevisiae Proteins , Simian Immunodeficiency Virus , Amino Acid Sequence , Circular Dichroism , Furin , Gene Products, env/biosynthesis , Gene Products, env/metabolism , HIV Envelope Protein gp160/biosynthesis , HIV Envelope Protein gp160/metabolism , HIV-1/metabolism , Humans , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Protein Precursors/biosynthesis , Protein Precursors/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Simian Immunodeficiency Virus/metabolism , Subtilisins/metabolism , env Gene Products, Human Immunodeficiency VirusABSTRACT
Leiurotoxin I (Lei-NH2), a toxin isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, is a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. It is a 31-residue polypeptide cross-linked by three disulfide bridges which are presumably between Cys3-Cys21, Cys8-Cys26, and Cys12-Cys28. To investigate the role of these disulfides, analogs of Lei-NH2 lacking one disulfide bridge (i.e., [Abu3,21]Lei-NH2, [Abu8,26]Lei-NH2, and [Abu12,28]Lei-NH2) were chemically synthesized by selective replacement of each pair of half-cystines forming a bridge by two alpha-aminobutyrate (Abu) residues. The two disulfide pairings of the main folded form of the synthetic analogs were established by enzymatic proteolysis. They were as expected between Cys8-Cys26 and Cys12-Cys28 for [Abu3,21]Lei-NH2 but were unexpectedly between Cys3-Cys12 and Cys21-Cys28 for [Abu8,26]Lei-NH2 and between Cys3-Cys8 and Cys21-Cys26 for [Abu12,28]Lei-NH2. The synthetic peptides were tested in vitro for their capacity to compete with the binding of [125I]apamin to rat brain synaptosomes and in vivo for their neurotoxicity in mice. In both assays, [Abu3,21]Lei-NH2 exhibited full Lei-NH2-like activity whereas [Abu8,26]Lei-NH2 and [Abu12,28]-Lei-NH2 possessed only residual activities (< 2% native toxin activity). This suggests that disulfide bridge Cys3-Cys21 is not essential per se for high toxin activity. Circular dichroism (CD) spectroscopy of the three analogs showed that only [Abu3,21]Lei-NH2 exhibited a CD spectrum similar to that of Lei-NH2, suggesting they both adopt closely related conformations, in agreement with the pharmacological data. Structural models of the analogs were constructed on the basis of the disulfide pairing assignment and compared with that of Lei-NH2.
Subject(s)
Disulfides/chemistry , Scorpion Venoms/chemistry , Amino Acid Sequence , Animals , Circular Dichroism , In Vitro Techniques , Iodine Radioisotopes , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Neurotoxins/metabolism , Protein Conformation , Radioligand Assay , Rats , Scorpion Venoms/chemical synthesis , Scorpion Venoms/metabolism , Scorpion Venoms/pharmacology , Synaptosomes/drug effects , Synaptosomes/metabolismABSTRACT
A DNA fragment has been isolated from the genome of Mycoplasma pirum by use of a genetic probe derived from the conserved region within the genes for the major adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. A gene encoding an adhesin-like polypeptide was localized, and sequence analysis indicated a G + C content of only 28%, with A- and T-rich codons being preferentially used. A total of 91% of positions 3 were either A or T. The deduced polypeptide is 1,144 amino acids long (126 kDa) and shows 26% identity with the adhesins of M. genitalium and M. pneumoniae. Other features in common with these two membrane proteins include a similar hydropathic profile and a proline-rich C terminus. Antibodies were prepared by using as an immunogen a peptide derived from the C terminus of the M. pirum adhesin-like polypeptide and were found to recognize on immunoblots a 126-kDa polypeptide from an M. pirum cellular extract. The characterization of the adhesin-like gene is a first step toward a better understanding of the mechanisms allowing this human mycoplasma to attach to host cells.
