Sequencing of proteins and peptides

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The amino acid sequence analysis offered by BioCentrum is performed by chemical sequencing or by MS method. Determination of amino acid sequence of the entire protein by chemical sequencing is more reliable than MS, however since it is very cost ineffective and time consuming for large proteins (more than about 350 amino acids) it is generally recommended only form smaller proteins (less than 300 amino acids) while large ones are analysed by MS.

Protein sequencing by Edman degradation
The samples (proteins or peptides) are subjected to sequential, so called Edman degradation cycles, realized by fully automatic apparatus (protein sequencer Applied Biosystems model 491 automatic sequencer). Using a special reagent, phenylisothiocyanate (PITC), the instrument sequentially detaches single amino acid residue from N-terminus of the analyzed polypeptide chain, convert it to phenylthiohydantoin (PTH) derivative and identifies it by HPLC chromatography.
Maximally, 30 to 40 amino acid residues can be identified from each analyzed sample. In case of dirty or non-homogenous proteins or peptides the Tris-Tricine SDS-PAGE electrophoresis is performed and the single protein stripes are electrotransfered onto a PVDF membrane, cut-out and subjected to sequencing.
Typically, the automatic sequencer is able to identify 19 proteinaceous amino acids (Asn, Asp, Gln, Glu, Ser, Gly, His, Arg, Thr, Ala, Pro, Tyr, Val, Met, Ile, Leu, Phe, Lys, Trp). Identification of Cys is possible only after previous derivatization with alkylating agents (such as iodoacetamide, vinylpyridine or acrylamide). Such derivatization is performed on a special demand, before sequencing, on a initial protein or peptide sample.
In case of longer (above 40 amino acids)  polypeptides  we offer protein fragmentation and sequencing of isolated internal peptide/peptides. Fragmentation of polypeptides can be done by cyanogens bromide or by enzymatic digestion by Arc-C peptidases or trypsin.

Protein sequencing by MS analysis – peptide mapping   
BioCentrum offers also the ability to identify unknown proteins using mass spectrometry techniques LC/MS. Obtained results are subjected to analysis using Mascot bioinformatics databases. This method is often used for the verification of known amino acid sequence as compared to a theoretical sequence. This method is also useful for determination of site of glycosylation, disulfide bridge mapping, methionine oxidation, etc.


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Important information considering protein sequencing