Protein Sequencing

Automated protein sequencing has evolved considerably with greater sensitivity, speed and ease of operation. Advances in mass spectrometry have now taken the center stage for protein identification. MS provides high throughput automation with more precise and powerful protein analysis. However, N-terminal sequencing by Edman degradation still continues to complement MS in difficult protein identifications. Currently, amino acid sequence analysis is performed on an Applied Biosystems Model 492 Procise Sequencer attached to a Model 140C Micro-gradient System and a 610A Data Analysis System.

Sample preparation

Samples for sequencing should be free of primary amines (e.g., Tris or glycine) and should contain less than 2 µmol equivalent of non-volatile salt (e.g., 100 µl of 20 mM NaCl). Ethanol-precipitation, trichloroacetic acid precipitation, reverse-phase chromatography, gel filtration, ion-exchange separation, Centri-Plus concentrator or dialysis can remove these materials. Chromatography and SDS-PAGE are two commonly used approaches for sample preparation. SDS-PAGE followed by electrobloting of protein samples onto PVDF membranes is the preferred method for N-terminal sequence analysis. PVDF membranes, pore size 0.2 micron, are known to have a superior protein binding capacity. To avoid high glycine background during Edman degradation, electroblotting must be performed in 10 mM CAPS (3-[cyclohexylamino-1-propanesulfonic acid]) buffer with 10% methanol, pH 11.0 for atleast 15- 30 minutes for 30 KDa size protein. Following the transfer, rinse the membrane 4-5 times with Milli-Q water to reduce the left over Tris and glycine contaminats from electrophoresis.

Staining

The proteins can be visualized on the blot by staining with Coomassie blue (originally developed as acid wool dyes in Kumasi, Ghana in the late 1800’s), Ponceau S, or Amido Black (the latter two are less sensitive stains). Coomassie blue is sensitive enough to detect 50-200 ng of protein on PVDF. PVDF-based membranes have higher protein-binding capacity and result in better average initial and repetitive yields (please note that nitrocellulose membranes are not compatible with the reagents and organic solvents used in automated protein sequencing). The size of an excised piece of PVDF membrane containing the protein band should be smaller than 40 mm2. As a rule of thumb, if the Coomassie blue stained protein band on the blot is visible on a photocopy, then there is enough material for sequencing. If necessary, a standard protein can be blotted on the PVDF to verify that the protein was electroblotted efficiently. Contaminating salts can be removed by washing the membrane with MilliQ water, prior to sequencing the sample on PVDF membrane. ProSorb cartridge, a device specifically designed for samples in solution containing salts, will be provided on request.

Protein sample in solutions

A protein sample may also be submitted dissolved in 25 µl or less of a suitable solvent, such as MilliQ water, 0.1% to 50% trifluoroacetic acid, 5% acetic acid, or aqueous acetonitrile. Sodium dodecylsulfate (SDS) at a concentration of 0.01% to 0.05% in a volume less than 100 µl may be used, if necessary. Samples that are submitted dry will be dissolved in 70% trifluoroacetic acid, unless the user specifies otherwise, and applied to the sequencer. The Core Facility cannot accept responsibility if later tests prove that this solvent was not effective for dissolving that particular protein or peptide.

Quantity of protein sample needed for sequencing. In general, 5 pmol or more of protein or peptide is required to obtain a reliable sequence. In majority of the cases, however, purity of the sample, rather than the amount, is the determining factor for successful sequencing. For example, pure and cleanly handled peptides from reversephase HPLC separation can be sequenced at 1-2 pmol levels. Whenever a sample protein does not give a readable amino acid sequence, 5 pmol of standard protein will be added to the sample disk, and sequencing run will be resumed. This procedure will verify if the sequencer is operating properly at a sufficient level of sensitivity. Turnaround time is generally three days.

It must be noted that more than half of all eukaryotic intracellular proteins have blocked amino-termini and cannot be sequenced directly. Artificial blocking of the amino-terminus may also occur during purification. The blocking group must be removed (if possible) before sequencing. Alternatively, the N-blocked protein can be identified by mass spectrometry. The N-blocked protein can be digested in-gel with trypsin or any other endopeptidase. The resulting peptides are then extracted from the gel and can be identified in MS by peptide mass mapping. The HPLC purified peptide fragments can also be sequenced to obtain the internal amino acid sequence.

Quantity of protein sample needed for sequencing

In general, 2 pmol or more of protein or peptide is required to obtain a reliable sequence. In majority of the cases, however, purity of the sample, rather than the amount, is the determining factor for successful sequencing. For example, pure and cleanly handled peptides from reverse-phase HPLC separation can be sequenced at 1-2 pmol levels. Whenever a sample protein does not give a readable amino acid sequence, 5 pmol of standard protein will be added to the sample disk, and sequencing run will be resumed. This procedure will verify if the sequencer is operating properly at a sufficient level of sensitivity. Turnaround time is generally three days.