Sequencing attomoles of peptide/protein: Automated methodology based on nanoscale HPLC and CE separations coupled to ion trap mass spectrometry through a nanoelectrospray ionization source

Currently, mass spectrometry is the method of choice for sequencing peptides/proteins. Unlike other methods, including Edman degradation, mass spectrometry is not limited by N-terminal blocking groups and allows unambiguous identification of unnatural and post-transitionally modified amino acids. Remaining challenges for mass spectrometry in sequencing biologically derived peptides include the complexity of the biological mixture and low peptide concentrations.;Recently there has been a lot of research toward the development of smaller sheathless electrospray ionization (n-ESI) sources to interface HPLC and CE separations to mass spectrometry. The overall effect of these n-ESI sources is an increase in S/N with less sample consumption. Current n-ESI designs have tended to be difficult to implement and are not amenable to automation. The work presented here is toward the development of novel liquid junctions for coupling CE and HPLC with n-ESI overcoming chemical noise problems and stability issues while providing detection limits of <100 amol. Finally, a n-ESI source design is described for interfacing n-HPLC and MS giving detection limits of <50 amol with automation capabilities.