Proteomics

Edman Protein Sequencing Services

Proteins and peptides are sequenced using our ABI 492 Procise sequencer. The sequencer can analyze samples containing 2-500 pmoles, either in solution or on PVDF. The number of cycles that can be clearly called depends on the quantity and purity of the sample, the number of proline residues and the presence of acid labile peptide bonds, especially Asp-Pro bonds. A pure, well-behaved protein present at 20 pmol or more can be sequenced for at least 50 cycles.

The ideal sample is a very clean protein or peptide, either in a solution with totally volatile solvents, or dried onto PVDF. If dried down or solublized samples contain non-volatile salts or detergents, they can be applied to devices called ProSorbs. These devices contain small discs of PVDF above a wick. The sample is applied to top of the device, and the solution is drawn through, causing the protein to adhere to the PVDF and the contaminants to filter through. The PVDF is then washed, leaving a clean sample, ready for sequencing. Electroblotted samples from SDS electrophoresis or isoelectric focusing can also be easily sequenced. It is suggested that electroblotting be performed using 10 mM CAPS, pH 11, 10 % methanol, rather than tris-glycine or tricine buffers. While these latter buffers can be used, they usually cause enormous contaminant peaks to appear on the first few cycles. Nitrocellulose cannot be used for blotting; it dissolves in the high acidity of the sequencer chemistry. If blotting peptides, it is best to use a type of PVDF with small pores designed especially for sequencing, such as Millipore PSQ or ABI Problot. Coomassie blue, amido black or Ponceau stains do not interfere with sequencing. Silver staining can be used for sequencing samples, but only with some modifications of the staining procedure. In general, if a band can be easily visualized with Coomassie, amido black or Ponceau, then there is sufficient quantity for sequencing. If a sample can be visualized by silver staining but not with Coomassie, then it probably cannot be sequenced successfully.

Sequencing is most successful if samples are pure and as clean as possible. During the last couple of steps of purification, it is best to wear powder free gloves, to protect samples from dust, and to use very clean glassware or good quality microfuge tubes. Samples that are not completely pure can be sequenced, but only if the desired protein constitutes about 80 % of the sample.

Customers should be aware that not all samples will yield a sequence. This may be because of low abundance or because the N-terminus is blocked. N-terminal amino acids may be blocked because an acetyl, formyl, carbohydrate or other group is attached to the ­NH2 on the first amino acid or because the N-terminal amino acid is a glutamine which has cyclized to form pyroglutamic acid. Approximately three-quarters of all mammalian proteins are N-terminally blocked; peptides and proteins from lower organisms are somewhat less likely to be blocked. There are no techniques which remove all types of blocking groups. There is an enzyme which will remove acetylated serines and threonines and another enzyme which will open pyroglutamate rings; however, use of these techniques requires a lot of guess work and a lot of sample. The most commonly utilized approach for dealing with blocked proteins is digestion with endoproteases, such as trypsin or LysLC. The resulting peptide fragments are then isolated by HPLC and sequenced. Digestion can be performed on proteins in solution or on bands in SDS electrophoresis gels or PVDF blots. The procedures work quite reliably when starting with 60 pmol of protein or more; however, they have been successfully performed with as little as 10 pmol protein. These procedures can be done in most labs that contain an analytical HPLC; we will be glad to provide references and procedures for digestion methods. Alternatively, we can perform these methods for you.

Samples can normally be applied to the sequencer within a day of receipt, and data can be e-mailed or faxed to the customer by the following day.

Sample Submission Form