Rapid Peptide Mapping and Protein Analysis by HPLC

Abstract

Peptide mapping by reversed phase high performance liquid chromatography (HPLC) has become a powerful technique that is widely applied in the structural analysis of proteins. Current methods employ columns packed with 5 to 10 µm macroporous alkyl silica sorbents and gradient elution with increasing organic solvent concentration in the eluent. Recent efforts in further development of the technique are aimed at increasing its sensitivity (Kalghatgi et al 1988, Lu et al 1986, Stone et al, 1986, Wilson et al 1986). Peptide mapping is also employed for analysis of purity and quality control of proteins produced by r-DNA technology and thus, finds applications in routine industrial analyses (Hancock 1986; Borman 1987). Recent reports from our laboratory described the use of short columns packed with 2 µm micropellicular stationary phase for rapid analysis of proteins and peptides at elevated column temperature (Kalghatgi et al 1987,1988, Maa et al 1988). As a result, the analysis can be completed in a few minutes or even in seconds. The high speed and efficiency thus obtained is associated with the pellicular configuration of the stationary phase, which allows rapid mass transfer of the eluate between the mobile phase and the sorbent, and to the use of elevated temperature which permits relatively high flow velocities of the mobile phase due to its lower viscosity. In this report we highlight main features of this approach based on the use of micropellicular reversed phase columns at elevated temperature to rapid HPLC that requires much shorter analysis times than the conventional techniques and expand its applications in protein analysis and peptide mapping.