Fractionation of Complex Protein Mixture by Virtual Three-Dimensional Liquid Chromatography Based on Combined pH and Salt Steps Zhi-Bin Ning Qing-Run Li Jie Dai Rong-Xia Li Chia-Hui Shieh Rong Zeng 10.1021/pr800318j.s002 https://acs.figshare.com/articles/journal_contribution/Fractionation_of_Complex_Protein_Mixture_by_Virtual_Three_Dimensional_Liquid_Chromatography_Based_on_Combined_pH_and_Salt_Steps/2910163 The complexity and diversity of biological samples in proteomics require intensive fractionation ahead of mass spectrometry identification. This work developed a chromatographic method called virtual three-dimensional chromatography to fractionate complex protein mixtures. By alternate elution with different pHs and salt concentrations, we implemented pH and salt steps by turns on a single strong cation exchange column to fully exploit its chromatographic ability. Given standard proteins that were not resolved solely by pH or salt gradient elution could be successfully separated using this combined mode. With a reversed phase column tandem connected behind, we further fractionated as well as desalted proteins as the third dimension. This present strategy could readily be adapted with respect to special complexity of biological samples. Crude plasma without depleting high abundance proteins were fractionated by this three-dimensional mode and then analyzed by reversed phase liquid chromatography coupled with LTQ mass spectrometry. In total, 1933 protein groups with wide dynamic ranges were identified from a single experiment. Some characteristics that correlated to the behavior of proteins on strong cation exchange columns are also discussed. 2008-10-03 00:00:00 LTQ mass spectrometry pH protein mixtures 1933 protein groups salt gradient elution Salt StepsThe complexity abundance proteins cation exchange column salt concentrations phase column tandem Complex Protein Mixture Crude plasma mass spectrometry identification salt steps cation exchange columns desalted proteins