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The goal of this workshop was to critically discuss several different procedures that enable internal sequences to be obtained from proteins isolated by SDS PAGE. The procedures discussed included enzymatic digestion following electroelution or passive elution from SDS polyacrylamide gels; in situ digestion of electroblotted proteins; elution of electroblotted proteins prior to digestion; and in gel digestion.
Electroelution is the "classical" procedure used to isolate proteins from SDS polyacrylamide gels and often gives high protein recoveries. Prior to digesting electroeluted proteins, it is essential that the quantity of SDS in the sample be reduced to a level that no longer interferes with either enzymatic digestion or subsequent reverse phase HPLC (1). This can be accomplished by: inverse gradient HPLC (2), TCA or acetone precipitation prior to digestion (1) or, more recently, by the use of an enzyme such as endoproteinase LysC, which is relatively resistant to SDS, coupled with the use of a DEAE precolumn that is connected just prior to the reversed phase column (3).
One advantage of PVDF blotting is that it removes all free SDS and buffer salts and the blotted protein is compatible with direct NH2-terminal sequencing. In order to obtain internal sequences from PVDF-blotted proteins, there are several options that may be considered. One procedure, which Joe Fernandez (Rockefeller University) described, is to block all protein binding sites on the membrane with PVP-40 and then digest the Ponceau S stained protein in situ in the presence of 1% Triton-X 100, 10% acetonitrile, and 100 mM Tris-HCl (4). When 3.5 ug (45 pmol) PVDF-blotted transferrin was digested in situ with trypsin, the overall sequencing yield of one peptide was 20%. This approach is recommended by the Rockefeller University Protein Sequencing Facility and has produced internal amino acid sequences from 90% of the proteins they have analyzed (4).
Another procedure used to obtain internal sequences from PVDF-bound proteins is CnBr elution followed by tryptic digestion. In this approach, the Coomassie Blue or Ponceau S stained, PVDF-blotted protein is cleaved in situ with CnBr, which significantly increases the subsequent elution efficiency, prior to extracting the CnBr fragments with consecutive 40% acetonitrile and 0.05% TFA, 40% acetonitrile washes. Based on 11 "standard" proteins, the average elution efficiency from Immobilon-P is 75% (5). For membranes such as ProBlott, which have a higher affinity than Immobilon-P does for low molecular weight proteins, the elution efficiency was only 17.3 % . Hence, only Immobilon-P blotted proteins are amenable to this procedure. This approach has been used in the W.M. Keck Biotechnology Facility at Yale University on over 30 "unknown" proteins with an overall success rate of 96% (5).
Larry Ward, (Joint Protein Structure Laboratory at The Ludwig Institute for Cancer Research) described an alternative technique to remove the protein from the PVDF membrane prior to digestion. This technique uses 2% SDS, 1% Triton X- 100 to elute Coomassie Blue stained proteins with an efficiency that ranges from 57-78 %. The SDS and Triton are then removed by "inverse" gradient HPLC. During this step, intact proteins are retained on small pore (60-120 Angstrom) reversed phase HPLC packings at high concentrations of organic solvent. The SDS washes through the column and the protein is then eluted by an "inverse" gradient that involves decreasing the concentration of the organic solvent in 0.4% TFA. The overall recovery of peptides from this procedure is about 30 % as determined with a 20 ug sample of lactoglobulin (2).
A disadvantage inherent with any electroblotting approach is the losses incurred during transfer onto the PVDF membrane. Without optimizing blotting conditions for a given protein, average blotting efficiencies have been reported to be only about 30% (1,2). In order to eliminate these losses, in situ digestion in SDS polyacrylamide gels was discussed as a viable alternative to PVDF blotting. Hence, Larry Ward described procedures they use for in gel digestion. Basically, the excised protein band is washed in water for 24 hours (to minimize the SDS) prior to partially drying the gel and then rehydrating it in 100 ~1 100 mM NH4HCO3, 0.5mM CaCl2, 0.02% Tween containing trypsin at a 1:10 (trypsin:sample; w:w) ratio. The sample is digested at 37deg.C for 12 hours and the resulting peptides sequentially extracted for 4 hrs with 100111 each of the following: 1% TFA, 70% TFA (2x), and finally, TFA/acetonitrile (50:50) (2). A variation of this in gel approach has been used in the W.M. Keck Facility at Yale (1). Again, the SDS is minimized by washing the gel in 0.1 M NH4HCO3 for 24 hours. The protein is then reduced, alkylated and digested in situ with trypsin. Peptides are eluted from the gel by washing for 6 hours with 0.1 M NH4HCO3 followed by an overnight wash with 2 M urea, 0.1 M NH4HCO3. The eluted peptides are then subjected to reverse phase HPLC. Based on 26 "unknown" proteins, the overall success rate of this procedure was found to be 92.9%.
Finally, preliminary data presented on the Kawasaki in gel approach suggested that at least for BSA, the recovery of peptides using this procedure was about 84 % . In this approach, the gel pieces are soaked in 100 mM Tris-HCI (pH 9.0) containing 0.1% SDS (37deg.C, 1 hr) prior to enzymatic cleavage. After cleavage, the gel is forced through a nylon mesh and the peptides are extracted by a 10-fold volume excess of 100 mM Tris-HCI (pH 9.0) containing 0.1% SDS. The peptides are separated using a reverse phase HPLC column with a DEAE precolumn. The DEAE precolumn removes the SDS and Coomassie Blue prior to the reverse phase HPLC (3).
In terms of comparing some of these approaches, the data that is available (that is based on large numbers of "unknown" proteins) suggests that the overall peptide recovery from the in gel approach may be as much as 3-fold above that of the CnBr elution approach, (Table I).
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Hence, the available data favors the in gel approach. For comparison, this table also shows peptide yields obtained from a large scale tryptic digest carried out under "ideal" conditions.
One of the conclusions reached during the workshop was that if the same sample was to be used for both NH2-terminal and internal sequencing, it would be easier to utilize an approach involving PVDF blotting. However, if the protein is assumed to have a blocked NH2-terminus (which is a reasonable assumption if it is from a higher eukaryotic source (6)) then higher peptide recoveries are likely to be obtained from an in gel approach. Lastly, the encouraging preliminary data on the Kawasaki procedure (3) suggests that comparative studies need to be carried out to determine if this approach might actually represent the best alternative.
References:
1. Stone, K.L., and Williams, K.R. (in press) in A Practical Guide to Protein and Peptide Purification for Microsequencing Vol 11 (Matsudaira, P. ed). Academic Press, New York.
2. Ward, L.D., Reid, G.E., Moritz, R.L., and Simpson, R.J.(1990) J. Chrom. 519: 199-216.
3. Kawasaki, H., Emori, Y., and Suzuki, K. (1990) Anal. Biochem. 191: 332-336.
4. Fernandez, J., DeMott, M., Atherton, D., and Mische, S. (1992) Anal. Biochem. 201: 255-264.
5. Stone, K.L., McNulty, D.E., LoPresti, M.L., Crawford, J.M., DeAngelis, R., and Williams, K.R., (1992) in Techniques in Protein Chemistry IlI(Angeletti, R. ed.) pp. 2334, Academic Press, New York.
6. Driessen, H.P., de Jong, W.W., Tesser, G.l. and Bloemendal, H. (1985) in Critical Reviews in Biochemistry (Fasman, G.D.,ed) Vol. 18, pp. 281-325, CRC Press, Inc., Boca Raton.
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