FAQs for N-Terminal Sequencing
Q1) What should I consider when preparing my sample for N-terminal sequencing?
One of the most important criteria in assessing the quality of a sample you are planning to submit for sequence analysis is the sample purity. Sample impurity is rarely the cause of total sequence failure. However, when an impure protein, with the presence of other contaminating proteins is used, the quality of the sequence information and length of read obtained will be reduced.
This is because the contaminating proteins will make it difficult to accurately quantify your sample concentration, which often lead to samples falling short of the usual yield criteria, making it difficult to evaluate the performance of the protein sequencer. As a result, these sequences are more prone to inaccuracies and the identifications are made with a lower degree of confidence than with a pure protein.
Thus, it is always better to minimize impurities and attempt to start a sequencing run with a chemically pure, homogenous sample.
The best way to determine sample purity is to employ multiple dimensions of chromatographic and electrophoretic analysis. If a protein sample runs as a single peak on both reverse phase HPLC and capillary electrophoresis it is reasonable to assume that it is pure.
However if the protein is purified by reverse phase HPLC, then reverse phase chromatography should not be used as a verification of purity. Capillary electrophoresis, SDS PAGE, or isoelectric focusing would be more appropriate techniques in this case. Demonstration of purity involves the accumulation of various purification analyses.
The more independent techniques you employ to demonstrate the purity of your sample the greater confidence you will have in its purity and the lower the risk of an unsuccessful sequence.
For a pure protein the sensitivity limit is approximately 1 picomole, however we recommend 10pmol to ensure a satisfactory result.
Insufficient material is one of the most common reasons in protein sequencing failures. Many times, the samples we receive contain far less amount of protein than estimated. This could be due to inaccurate estimations of the yield of the protein after each step of their protocol without any actual assays, and even the use of unreliable methods in protein quantitation assays. This often leads to samples that are sequenced but do not contain sufficient protein to give a detectable sequence.
The way to avoid this problem is to actually determine the amount of protein in or on the material you submitted for sequencing. Dye binding assays (Lowery, Coomassie stain, BioRad, etc.), where the protein concentration is estimated relative to the response obtained cannot be done accurately with a standard solution of some unrelated protein and can result in very large errors. Experience has shown that the most reliable method would be amino acid analysis.
It can be performed on all sequencing samples if they are submitted in an appropriate matrix, e.g. suitable buffer solution, PVDF, Sequelon, etc. The information thus obtained can then be used together with the analysis of an appropriate blank and an estimate of molecular weight to accurately determine the amount of protein in your sample.
Blocked Amino Terminus:
This problem is frequently encountered and results in the lack of any observed sequence. Furthermore it is impossible to differentiate this situation from a case of insufficient material. Both problems result in blank cycle chromatograms and no observed sequence. However, if your sample was well analyzed to indicate its purity and quantity of protein, it can be reasonable to conclude that your protein is blocked. In nature the N-terminal residue of a protein can be subject to post-translational modification. This is a biological process that occurs in the cell, and is not normally one due to sample preparation. CommonN-terminal blocking groups include pyroglutamate, N-acetyl groups such as acetyl¬serine or acetyl-threonine and N-formylated amino acids. NOTE: More than 50% of all eukaryote proteins are blocked.
If the protein is blocked you must try to determine if it was blocked naturally or if it was blocked during the isolation process. If the blocked amino terminus is an artefact of the isolation protocol it is best to determine the step or reagent responsible and either modify or eliminate it. If the protein is blocked naturally then you will have to attempt an internal sequence. This cannot be predicted prior to analysis, and consequently the no result fee will apply.
NOTE: In such cases the best alternative to determine the protein identity is using mass spectrometry [Service 001]. Whilst this will not determine the N-terminal sequence of a blocked protein it can be used to unequivocally identify the protein. New samples would be required in the form of gel bands.
The protein sample should be free of interfering substances. These are defined as non-protein chemical contaminants that can inhibit the sequencing reaction, modify the sequencing conditions or interfere with the interpretation of results. They include primary and secondary amines (glycine, Tris, etc.), many detergents (Triton, SDS etc.), UV absorbing materials, high concentrations of non-volatile buffering salts (phosphate, citrate etc.).
These substances can be easily removed from sequencing samples by binding the protein to a PVDF membrane and washing the material thoroughly to remove all of the interfering material.
It is important to bear in mind that protein sequencing is expensive and when you submit a sample for sequence analysis you will be charged for this service whether or not a sequence is obtained. We will terminate bad sequencing runs as soon as possible to minimize the cost of these failures. However, the quality of the sample submitted for sequencing is the responsibility of the party submitting the sample.
Q2) How should I submit my sample for N-terminal sequencing?
Ans: When purifying your protein by gel electrophoresis it is advised to isolate it from the gel matrix and other interfering compounds by electroblotting to PVDF, as it is the only membrane material sturdy enough to withstand the protein sequencing process. These membranes are available from Bio-Rad, Applied Biosystems, GE, and others. Blots must be free of any particulate material. Other common membrane materials such as nylon or nitrocellulose are not suitable. Multiple bands of PVDF can be sequenced simultaneously, up to a maximum of ten to twelve 2x5 mm pieces of PVDF can be placed in the sequencer cartridge without inhibiting reagent flow.
Do take note of the following
- Protein band must be visible by Coomassie Blue staining.
- The PVDF membrane should be sent intact, along with a photocopy indicating the band to be sequenced. We would excise the band for you.
Q3) Which is a good membrane to use?
Ans: SequiBlot PVDF Membrane for Protein sequencing (0.2μm) [BioRad Cat# 162-0182].
Q4) The sample is in a gel, can it be sequenced?
Ans: No, it must be transferred to sequencing grade PVDF membrane.
Q5) Can samples be analysed in liquid form?
Ans: Yes, providing the sample is pure and in a low salt buffer e.g. 50mM ammonium hydrogen carbonate; for TRIS or phosphate buffers the maximum salt strength is 20mM. NaCl, surfactants (e.g. SDS) and glycine cannot be present. The sample must be freeze dried or lyophilised in a micro-centrifuge tube prior to shipping.
NOTE: The type of buffer and the volume of the liquid before drying must be stated on the Sample Form.
NOTE: Complex samples containing more than one protein cannot be analysed by this process
Q6) How much material do I need for N-terminal sequencing?
Ans: We recommend that you provide us with at least 10 pmoles of material. It is important that you estimate the concentration of your liquid samples and the amount of protein bound to your PVDF samples accurately. The most effective way to do this is by amino acid analysis. It is preferable to load at least 10 picomoles of protein/peptide for each sequence but we can work with less. Useful sequence information can be obtained with as little as 0.5 pmoles; however, the probability of sequencing success is directly proportional to the amount of protein/peptide used.
Q7) If I just want to identify the protein from a database, how many residues should I sequence for?
Ans:We recommend fifteen residues for a database search. Search engines usually need 8-12 residues, but the first few cycles of sequencing can be uncertain due to high background. This helps to ensure your chances of identifying your protein of interest and minimize the risk of an ambiguous search.
Q8) Is cysteine a problem?
Ans: Yes, Cys can only be detected on the sequencer following special alkylation with 4-vinylpyridine. This is only possible with liquid samples and must be requested before analysis commences (additional charge apply).
NOTE: If Cys is not derivitised then it cannot be detected under normal conditions and its presence is inferred by a gap in the sequence, reported as X.
Q9) Can proteins be unblocked?
Ans: Deblocking of proteins is rarely possible and not available. Unfortunately, no further analysis of blocked samples is possible.