Online dual gradient reversed-phase/porous graphitized carbon nanoHPLC for proteomic applications.

The analysis of proteolytic peptide mixtures is among the dominant tasks within proteomic workflows. In order to limit undersampling effects during mass spectrometric detection, online-coupled liquid chromatography is the method of choice, with reversed-phase chromatography being the most important separation mode. Since hydrophilic compounds such as short peptides and some glycosylated species as well as oligosaccharides from glycoproteomic workflows are commonly not accessible by this analytical setup, we hereby present a dual gradient system combining reversed-phase and porous graphitic carbon retention modes within a single nanoHPLC setup. Samples in the low femtomole range are analyzed consecutively first by reversed phase, and nonretained molecules are directly separated by porous graphitic carbon. Both gradient elution systems allow for online coupled mass spectrometric detection and are demonstrated to enable analysis of protein, peptide, and oligosaccharide mixtures within the same setup. Thereby, the accessible range for proteomic and glycoproteomic applications may be extended far beyond the limits of conventional reversed-phase nanoHPLC setups.