Reverse-Phase HPLC Separation of Enzymatic Digests of Proteins

Abstract

Reverse-phase HPLC is a powerful technique having the ability to resolve complex mixtures of peptides within a short amount of time, in volatile solvents and is compatible with mass spectrometric approaches. Recent technological advances in HPLC analysis include improved stationary phase packing materials for columns (moving from 10 μm to 3 μm and now 1.7 μm particle sizes) and next generation LC systems that have improved resolution, speed and sensitivity and operate at ultra high pressures of up to 10,000 psi (1). These LC systems are commonly referred to as ultra high pressure or ultra performance liquid chro-matography (UPLC). In general, we find that peptides that are less than about 30 residues in length usually separate based on their content of hydrophobic amino acids and that their relative elution positions can be reasonably accurately predicted from published retention coefficients (2, 3). Since proteins often retain some degree of folding under the conditions used for reverse-phase HPLC, the more relevant parameter in this instance is probably surface rather than total hydrophobicity. Although larger peptides and proteins may be separated on HPLC, sometimes their tight binding, slow kinetics of release, propensity to aggregate, and relative insolubility in the usual acetonitrile/0.05% trifluoroacetic acid mobile phase results in broad peaks and/or carryover to successive chroma-tograms. In our experience, these problems are seldom seen with peptides that are less than about 30 residues in length, which thus makes reverse-phase HPLC an ideal method for fractionating enzymatic digests of proteins. Although it is sometimes possible to improve a particular separation by lessening the gradient slope in that region of the chromatogram, generally, enzymatic digests from a wide variety of proteins can be reasonably well fractionated using a single gradient that might extend over 1–2 h. Another advantage of reverse-phase HPLC is its excellent reproducibility which greatly facilitates using comparative HPLC peptide mapping to detect subtle alterations between otherwise identical proteins. Applications of this approach might include identifying point mutations as well as sites of chemical and posttranslational modification and demonstrating pre-cursor/ product relationships. Finally, since peptides are isolated from reversephase HPLC in aqueous mixtures of acetonitrile and 0.05% to 0.1% of an acid (typically TFA, acetic acid, or formic acid) they are ideally suited for subsequent analysis by matrix-assisted laser desorption mass spectrometry (MALDI-MS, 10), on-line LC-MS/MS and automated Edman sequencing.