Establishment Of Three-dimensional HPLC-MS/MS System For Complex Proteome Fractionation

The sufficient separation of protein and peptide mixture is important for the highsensitivity of proteome identification in proteomics research. Multi-dimensionalliquid chromatography (MDLC) combines various chromatography types to increasethe capacity of separation system greatly, satisfying the demand of high efficiency,high reproducibility and high automation of sample fractionation. The most widelyused MDLC approach is two-dimensional liquid chromatography (2DLC) with thecombination of ion-exchange chromatography (IEX) and reversed phase (RP)chromatography to separate peptides digested from complex samples. Recently, morepowerful three-dimensional liquid chromatography (3DLC) systems have beendeveloped through the addition of one-dimensional chromatography prior to theclassic2DLC, and they have revealed the potential of efficient simplification ofcomplex proteome samples.In this study, two3DLC fractionation systems were established, combining thetraditional2DLC with peptide pre-fractionation and protein pre-fractionationrespectively. As an example of complex proteome sample, cell lysate of mouse breasttumor cell line4T1was fractionated and further detected with mass spectrometry. Inone typical experiment, about1,200proteins were identified via traditional2DLC andlater MS detection and data processing. Through peptide pre-fractionation based3DLC method, about2,500proteins were identified, demonstrating that theenhancement of separation dimension reduced the sample complexity and increasedthe protein identification ability. Moreover, through the protein pre-fractionationbased3DLC method, a higher number of about3,000proteins were identified withproteins, especially high abundant ones, enriched more efficiently, indicating thatprotein pre-fractionation based3DLC method is more suitable for the fractionation ofhighly complex proteome sample.