Protein Sequence Analysis technical / background information
N-terminal sequencing, the generally accepted and most reliable method to determine the amino acid sequence (the primary structure) of a protein is performed by automated Edman degradation [1,2]. The automated equipment used are Applied Biosystems Model 494 HT Procise Protein Sequencing Systems . Proteins or peptides are sequentially broken down from the N-terminus and the derivatised residues are separated and identified by on-line coupled RP-HPLC.
N-terminal sequence analysis of 1-2 pmol protein or peptide, or even less, is possible. However, to get the best results and to avoid disappointments, a minimum quantity of 5-10 pmol is desirable, especially since only 20-80% of the protein material is sequenceable ('initial yield'). If available, higher quantities may be advantageous (e.g., to repeat the analysis with special sequence cycles to improve the results). Please take into account that the 'initial yield' of proteins electro-blotted onto PVDF after SDS-PAGE is usually lower (5-30%), and, therefore, at least 25 pmol of the protein of interest should be applied to the gel (the total area of the piece of PVDF containing the protein band should be as small as possible and should not exceed 0.8 cm2; see also our separate information "Sample preparation by SDS-PAGE and electro-blotting onto PVDF”.
Higher quantities of at least 200 pmol should be provided if the purpose of the analysis is (also) for purity reasons and/or batch release purpose, in order to allow detection of protein impurities to the 0.5 – 1.0% level.
Although we have a lot of experience in analyzing mixtures as well, for a reliable, unambiguous sequence assignment the protein samples should be as pure as possible and preferentially free of salts (Tris!), amines and excessive SDS. Salt-free samples are not strictly necessary, since we offer very effective pre-purification services as well.
Samples can be sent either dry (speed-vac or lyophilized), blotted onto PVDF or PCGM membranes, or in a piece of SDS-PAGE gel.
Routinely, 20 to 30 residues can be easily identified, even for PVDF samples. Difficulties can be encountered in identifying tryptophan residues, especially at low sequence level. In principle, Glycoproteins can be sequenced equally well, except that certain positions may result in a gap in the sequence analysis, because of the presence of a glycosylated residue, which are in most cases not observed. Cysteine and cystine cannot be identified as such, and, therefore, should be modified (alkylated) to a stable derivative (see Order form under Options). Identification of certain modified amino acids, e.g. carboxyamidomethylcystein, pyridylethylcystein, Ornithine, MetO2 and hydroxyproline is possible, as well as certain un-natural amino acids like AIB etc.
Standard sequence analysis includes database search and consultation. Standard delivery includes raw data, product information and a signed report containing the scientific interpretation of the results.
Approximately 1-2 weeks after acceptance of order.
(Pre-)purification / desalting methods, e.g., RP-HPLC, SDS-PAGE / PVDF blotting, or protein isolation from gel. Protein fragmentation procedures (chemically or enzymatically). Reduction and alkylation for positive identification of Cys residues. Checks for N-terminal blocked proteins including de-blocking procedures (formyl, acetyl, pyroglutamate. Internal sequence analysis (in case of blocked N-terminus). Extended sequence runs by using OPA-blocking at Proline residues. Identification of post-translational modifications. Sequencing of low radioactive samples. Complete sequence analysis. Computer sequence analysis. Specific isolation of the C-terminal peptide of proteins. C-terminal sequence analysis.
The minimum number of sequencing cycles (=amino acids) to be ordered is 5. The number of amino acids that will be charged equals the number of positions which resulted in interpretable sequence information (either unambiguous or tentative assignments). Usually, the customer will be consulted during the analysis about the number of degradation cycles to be performed in case (only) tentative assignments are expected. In order to get the best results, please fill in the sample information part of the order form.
We would be pleased to advise you on the best way of preparing your sample.