[Establishment of a microscale peptide prefractionation system based on ultra performance liquid chromatography and...

Liquid chromatography (LC) and mass spectrometry (MS)-based proteomics now allows very deep coverage of proteomes. Two-dimensional high performance liquid chromatography (2-D HPLC) is a useful tool for the proteome analysis of complex biosystem. However, it has drawback of a long running time, and it typically requires peptide amounts in the milligram range, and large volume of collected fractions. In this study, we introduce ultra-performance liquid chromatography (UPLC) and an eight-port rotor valve as a highly efficient and convenient method for a first-dimension separation and collection system. The combination of our UPLC-based fractionation using basic buffers with an online LC-MS/MS provided orthogonal peptide separation and demonstrated the powerful performance for protein identification. Upon applying the novel method to triplicate measurements of a human cell line, we observed excellent quantitative reproducibility between replicates (coefficient of determination R²>0.95) and more than 23.52% peptide identifications over the conventional StageTip approach. The fractionation method described here is flexible, straightforward, and robust, and it enables proteome analysis with minimal sample requirements.