Development of new methods to study the amino terminalpeptide of proteins and their applications in the biotechnological industry

Introduction The fast development of genetic engineering and biotechnology has made possible to obtain proteins naturally expressed at so low concentrations that it makes hard to be isolated. The recombinant DNA technology allows inserting a gene coding for a protein of interest into the genome of a host microorganism by means of an appropriate genetic construct. Then, the recombinant protein is obtained in sufficient amounts by growing the host microorganism, and its subcellular location can be predetermined to implement a successful purification strategy. However, during translation, expression, purification or storage processes, proteins can be chemically modified, such modifications altering their primary structure and their subsequent biological activities. Additionally, degradation processes mediated by exopeptidases can frequently modify their amino (Nterm) and carboxyl ends. One of the most common post-translational modifications is the blocking of the N-term group. It is estimated that over 80% of eukaryotic proteins are blocked in the N-term [1]. Such modification makes sequencing impossible by the Edman’s method. Therefore, the international regulatory agencies request the information of that end on the proteins to control the quality of recombinant proteins. Most of the methods alternatives to the Edman’s degradation method are based on mass spectrometry (MS) as analytical tool to study the proteins primary structure. They use a combination of enzyme digestion and/or chemical modification of primary amino groups preceded by a chromatographic step. Our work comprises three new methods to study the N-term peptide of proteins, whether blocked or not. One of them (named method 1) selectively isolates the N-term peptide, while the other two (methods 2 and 3, respectively) allow its identification in a MS spectrum, starting with the protein isolation from a gel band after fractioning and direct identification. Method 1 is optimized for proteins in solution and useful to study N-term-blocked (N-blocked) proteins, easily isolating the N-blocked peptide in a fast and effectively by successive digestions and a cationic exchange step (Figure 1). Nevertheless, this method is not so efficacious to study neither proteins expressed in low amounts nor to analyze proteins with free N-terms or in stability studies to identify degradation products. Thus, the other two methods (methods 2 and 3) were optimized to study proteins isolated from polyacrylamide gels, and therefore, in very low amounts. Both allow the identification of the N-term peptide among the other peptides in the MS spectrum following enzyme digestion, a significant difference with previously reported methods. This identification is made by means of the ESIMS spectrum analysis, identifying the N-term peptide according to the isotopic distribution. Method 2 makes the study of both N-blocked and N-free proteins possible, and together with method 3 they can be used to study protein degradation products, one of the main goals of stability studies (Figure 2). Method 3 combines the identification of the Nterm peptide in an ESI-MS spectrum with the ability to differentiate the fragmentation series in the ESIMS spectrum to achieve an casier and more reliable sequencing which is particularly advantageous when proteins are derived from organisms with unknown genome.