Automated tandem high-performance liquid chromatographic system for separation of extremely complex peptide mixtures

We have developed an automated tandem chromatography system, which consists of a combination of ion-exchange column chromatography and reversed-phase column chromatography. The system is composed of two independent high-performance liquid chromatography assemblies, in each of which programmed elution is carried out by a computer-assisted controller. A peptide mixture is applied to an ion-exchange column and is eluted in a stepwise manner. The eluent from the first column is introduced directly into the second, reversed-phase column, which is connected in tandem through a tee tube. After application of two column volumes of the eluent, reversed-phase chromatography is performed by linear gradient elution. Stepwise elution for ion-exchange chromatography and the gradient elution for reversed-phase chromatography are synchronized by a computer program. The resolving power and the reproducibility of the method were tested by using a tryptic digest of human ceruloplasmin [molecular weight 132 000 daltons (132 kDa)]. By this method, the digest was resolved reproducibly into several hundred peaks within 16 h. All of the four glycopeptides expected to be obtained by tryptic digestion were purified easily from the whole digest of the protein. Comparison of the peptide maps between a single-chain and a degraded form of ceruloplasmin facilitated the identification of two tryptic peptides, derived from the carboxyl-terminal regions of 67 kDa and 50 kDa fragments of the degraded form, which lack the carboxyl-terminal arginine and lysine residues, respectively. The method may be applicable to comparative peptide mapping of very large proteins exhibiting molecular microheterogeneity, such as carbohydrate or genetic variants; it also can be used complementarily for sequence support of DNA sequencing as well as for preparative purification of peptides as a strategy of protein sequencing of very large proteins.