Examination of automated polypeptide sequencing using standard phenyl isothiocyanate reagent and subpicomole high-performance liquid chromatographic analysis.
The feasibility of accurate protein sequencing at the subpicomole level, using automated Edman chemistry and "on-line" HPLC analysis, was studied. Several modifications of the standard system were first introduced. A larger portion of the phenylthiohydantoin amino acids (70%) is analyzed. Dissolution in 10% acetonitrile is improved by short periodic bursts of argon. Losses on the column of subpicomole amounts of analytes, in the presence and absence of scavengers, were quantitated; they are related to destruction rather than to unspecific sticking to the stationary phase. Baseline drift, for a large part caused by the presence of ultraviolet absorbing N,N-dimethylphenylthiourea in solvent B, is completely eliminated by the addition of a twofold molar excess of tryptophan to solvent A. This allows real time recording of the 269-nm absorption detector signal at 0.0005 absorption unit full scale. The combined modifications result in an eightfold increase in sensitivity over standard methods. Sequence calling at the 2 to 10 pmol level, through visual inspection of chromatograms, becomes increasingly simple this way. Once the sequenceable signal drops below the 1 pmol level in the course of a run, meticulous comparison and matching of the preliminary calls with a spreadsheet of peak integration data are necessary for accurate assignments. Reliable sequencing, with signals at the subpicomole level, is now feasible for stretches of over 10 residues. Contaminating amino acids and polypeptides and incompletely removed reaction by-products constitute a major problem for analysis at this level. Future limits to sensitivity of Edman sequencing will primarily depend on improved micropreparations of proteins in cleaner environments, higher purity reagents and solvents, instrument miniaturization, and solid-phase techniques.
Tempst
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P
Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115.
Riviere
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D000595:Amino Acid Sequence; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017879:Isothiocyanates; D007782:Lactoglobulins / Q000032:analysis; D008722:Methods; D008832:Microchemistry / Q000379:methods; D010455:Peptides / Q000032:analysis*; D010669:Phenylthiohydantoin; D010670:Phenylthiourea / Q000031:analogs & derivatives / Q000378:metabolism; D013861:Thiocyanates
D017879:Isothiocyanates; D007782:Lactoglobulins; D010455:Peptides; D013861:Thiocyanates; C005441:phenylisothiocyanate; D010669:Phenylthiohydantoin; D010670:Phenylthiourea; C005355:U 10157