MAKING PEPTIDE MAPSPeptide mapping is a workhorse technique in biopharmaceutical characterization.1 It is used to identify proteins based on the elution pattern of the peptide fragments, determine post-translational modifications, confirm genetic stability, and analyze protein sequence when interfaced with mass spectrometry.
To make a peptide map, it is necessary to separate every peptide into a single peak. Therefore, peptide mapping represents a significant chromatographic challenge, because of the complexity of peptide digests. In addition to the large number of peptides that are generated from the enzymatic digest of a protein, there can be a large number of alternative peptide structures, such as post-translational modifications.
There has been a long trend of reducing particle size in liquid chromatography. Modern reversed phase liquid chromatography (LC) began in the mid-1970s with the advent of irregular 10 μm particles, and within the last five years 2.5 μm particles have become available. The smaller particles have been used in short columns, which leads to fast analysis times but relatively modest gains in resolving power.
Column length has decreased with particle size because the system pressure required is inversely proportional to the cube of particle diameter. For example, reducing the particle size by a factor of two requires an increase in the operating pressure by a factor of eight. It is, therefore, necessary to use shorter columns at lower flow rates to remain within the capabilities of the instrument. Clearly, in order to take advantage of smaller particle sizes, both in terms of improved speed and improved resolution, we require instrumentation capable of high-pressure operation. In addition, system band broadening must be reduced to observe the narrow peaks generated with small particle packings.
In 2004, Schwartz and Murphy introduced the first LC system capable of operation up to 15,000 psi (1,000 bar).2 The combination of a system capable of high-pressure operation and columns packed with sub-2 μm particles has been termed ultra performance liquid chromatography (UPLC) to differentiate it from high performance LC (HPLC).3