Microbore reversed-phase high-performance liquid chromatographic purification of peptides for combined chemical sequencing-laser-desorption mass spectrometric analysis

Abstract

An optimized microbore RP-HPLC system (1.0 mm I.D. columns) for the purification of low picomole amounts ( < 5 pmol) of peptides is described. It is comprised of commercially available columns, instrument components and parts. These were selected on the basis of a comparative evaluation and to yield the highest resolution and most efficient peak collection. The sensitivity of this system equals, probably surpasses, that of advanced chemical microsequencing for which 2–4 pmol of peptide are minimally required. As an automated sequencer cannot be “on-line” connected with a micro-preparative HPLC system, fractions must be collected and transferred. With a typical flow of 30 μl, efficient manual collection is possible and fractions (about 20 μl in volume) can still be handled without unacceptable losses, albeit with great precaution. Furthermore, major difficulties were encountered to efficiently and quantitatively load low- or sub-picomole amounts of peptide mixtures onto the RP-HPLC column for separation. Discipline and rigorous adherence to sample handling protocols are thus on order when working at those levels of sensitivity. With adequate instrumentation and handling procedures in place, we demonstrate that low picomole amounts of peptides can now be routinely prepared for analysis by combined Edman-chemical sequencing-matrix-assisted laser-desorption mass spectrometry (MALDI-MS). The integrated method was applied to covalent structural characterization of minute quantities of a gel-purified protein of known biological function but unknown identity. The results allowed unambiguous identification and illustrated the power of MALDI-MS-aided interpretation of chemical sequencing data: accurate peptide masses were crucial for (i) confirmation of the results, (ii) deconvolution of mixed sequences, (iii) proposal of complete structures on the basis of partial sequences, and (iv) confirmation of protein identification (obtained by database search with a single, small stretch of peptide sequence) by “mass matching” of several more peptides with predicted proteolytic fragments.