Due to its importance in scientific research and in the quality control of synthetic peptides and/or recombinant proteins, the structural analysis of peptides and proteins constitutes one of the most relevant topics in biotechnology. Primm offers a wide range of advanced technical services which are very important for the structural characterization and development of bio-molecules, such as peptides and proteins. These services are based on the integration of classical biochemical techniques, such as N-terminal protein sequencing, with advanced mass spectrometry procedures.
The sequence of the first 10-20 residues at the N-terminal of a protein gives sufficient information to allow the following:
- Assigning the site of proteolytic maturation
- Synthesizing oligonucleotide probes for specific cDNA isolation
- Identifying the protein in databases
- Aligning the protein against DNA sequences
- Assessing the purity of the peptide/protein
Additionally, the determination of the accurate molecular weight of a peptide or a protein gives the following analytical information:
- definition of the purity of the peptide/protein
- quality control for peptide synthesis
- confirmation of the protein sequence
- control of the structure of recombinant proteins
- characterization of genetic variants or mutant proteins in which there is an amino acid substitution or modification
- definition of the proteolytic processing of the protein, both at the N- and C-termini
- identification of post-translational maturation events or chemically induced changes
- identification of the glycosylation pattern of glycoproteins.
The N-terminal sequence of peptides and/or proteins (5-20 residues) can be carried out on samples purified by HPLC or mono-and bidimensional SDS-PAGE, followed by electroblotting on PVDF (Polyvinylidene difluoride) membranes using the well known EDMAN degradation method. Pre-treatment of the sample, i.e. reduction and alkylation of the cysteine residues, is also performed.
The accurate molecular weight of peptides and proteins is determined by electrospray (ESMS) and MALDI-TOF mass spectrometry analysis of crude or HPLC purified samples. Pre-treatment of the samples and/or reverse-phase HPLC purification can also be performed. Identification of modified components or contaminating species is also provided.
Analysis of synthetic peptides
Mass spectrometric analysis of synthetic peptides using different techniques is carried out both as quality control on a "batch to batch" basis and to establish the structural identity of "custom synthesis" products. The characterization of bioactive peptides containing unusual structural features is also performed.
Characterization of genetic variants and mutants
The sites and nature of structural abnormalities occurring in protein variants are identified. Structure verification of mutants obtained by site-directed mutagenesis is also performed.
Identification of post-translational modifications
The assessment of the sites and nature of modifying groups originating from post biosynthetic events (methylation- phosphorylation-hydroxylation etc.) or the presence of proteolytic processing at both the N-and C- termini of proteins is carried out.
Identification of labeled residues
Chemically labeled amino acids, i.e. those occurring at the active site of enzymes or important for the biological activity of proteins are identified.
Definition of the oligosaccharide structure of glycoproteins
The definition of the entire structure of the oligosaccharide moieties released from glycoproteins is achieved by determining the microheterogeneity of the different structures, the composition of the monosaccharides present, the branching pattern, the sequence of the antennae, and the possible occurrence of modifying groups, such as sulphate, phosphate, acetyl groups etc. Furthermore, the linkage positions of the individual sugars in the oligosaccharide chains and the glycosylation sites in the protein sequence are also determined.
Amino acid sugar and lipid analysis
Unusual amino acids occurring in bioactive peptides, monosaccharides, and lipid components obtained from glyco-and lipo-proteins are identified by GC-MS following derivatization of the samples.
Proteins purified by HPLC or excised from a mono or bi-dimensional electrophoresis gel can be digested in vitro by proteolytic enzymes, such as trypsin, yielding a mixture of peptides generated on the basis of the presence of Arginine and Lysine residues in the original protein sequence. The peptide mixture is then analysed by MALDI -TOF mass spectrometry which gives the accurate molecular weight of some or most of the peptides. The comparison of the experimental molecular weights with those generated by virtual computer theoretical trypsin digestions of the protein sequences present in public databases, allows us to determine the nature of the protein sample and to precisely identify the protein target.
