Packed capillary perfusion columns were evaluated for their ability to rapidly separate and sequence enzyme digests by HPLC/MS/MS using electrospray ionization (ESI) and were compared directly against packed capillary (180-mu m and 320-mu m i.d.) and microbore (1.0-mm i.d.) reverse-phase materials (e.g., Vydac C18, 5-mu m, 300-Angstrom particles). The relative sensitivities when coupled directly to electrospray ionization were also compared. In order to ensure that the perfusion columns were operating under perfusing conditions, the Linear velocity of the 320-mu m-i.d. capillary perfusion columns was set to 3000 cm/h. This linear velocity translated roughly to a flow rate of 45 mu L/min. This enabled retention times to be reduced by a factor of 3-5 relative to those of capillary C18 180-mu m-i.d. and 320-mu m-i.d. columns. Separation efficiencies were comparable for the analysis of a src homology 2 (SH2) tryptic digest and an endoprotease Lys-C digestion of a phosphodiesterase PDE-IV protein. Separation of tryptic-digested human growth hormone proved more difficult by capillary perfusion chromatography than by capillary C18 column chromatography. This was attributed to the large number of relatively low molecular weight peptides (less than or equal to 1500 De) produced upon digestion. The feasibility of performing in-line LC/MS/MS for the sequence determination of enzymatic digests using capillary perfusion columns was also evaluated. Peak widths for peptides separated on 320-mu m-i.d. capillary perfusion columns were found, in general, to be 5-10 s at full width at half-maximum (FWHM) whereas the FWHMs for conventional capillary C18 columns were between 20 and 45 s. It was unclear whether or not in-line capillary perfusion LC/MS/MS would be capable of summing a sufficient number of scans to produce high-quality CID spectra for low picomole quantities of peptides. Tryptic-digested src homology (SH2) domain of pp(60c-src) and human growth hormone (hGH) provided suitable models for the in-line LC/MS/MS studies. Using a modified PE-Sciex API III collision cell it was possible to fully sequence several of the tryptic peptides of hGH from a 25-pmol on-column injection. The sensitivity of the perfusion LC/MS/MS experiment was compared directly against capillary C18 LC/MS/MS. The sensitivity of detection by C18 LC/MS/MS was approximately 20-fold better than that achieved using the perfusion column. This is consistent with reports by Covey et al. which illustrate that the greatest absolute electrospray sensitivities are achieved with the lowest flow rates (Covey et al. Application Note L-1650, PE-Sciex, Thornhill, ON, Canada). Preliminary results clearly demonstrated the feasibility and potential for routine and rapid separations and sequencing of peptides by capillary perfusion column LC/MS/MS at the 25-pmol level. Methods for improving the sensitivity of detection for perfusion column LC/MS and LC/MS/MS by IonSpray are proposed.
