Capillary hplc: A method for protein isolation and peptide mapping

Capillary or microcolumn (<0.5-mm internal diameter) HPLC is an extremely powerful microseparation technique for proteins and peptides. It has distinct advantages over wider-bore column HPLC, such as increased mass sensitivity and lower flow rates, which make it particularly attractive for coupling to hyphenated detection techniques such as mass spectrometry. We describe a procedure for enhancing conventional HPLC systems to provide the accurate low flow rates (0.4-4 μl/min) and gradients necessary to operate slurry-packed capillary columns. A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to a number of commercial UV detectors. Detailed procedures are given for the fabrication of ≤0.32-mm-internal-diameter polyimide-coated fused-silica columns, slurry-packed with reversed-phase chromatographic supports. The chromatographic performance of these columns is illustrated using a series of standard proteins and a mixture of peptides derived from a Staphylococcus aureus V8 protease digest of recombinant murine interleukin-6. We demonstrate that capillary HPLC is an effective method for separating and collecting peptides from one- and two-dimensional polyacrylamide gels, in quantities sufficient for obtaining amino acid sequence data using either Edman methods or mass spectrometry.