The Establishment Of One Minute Overall Protein Liquid Chromatography

The study on the separation of intact proteins in proteomics, protein drugs and the purification of proteins in native plants and animals need very fast separation by high-performance liquid chromatography (HPLC). Peter Carr's group suggested that one-dimensional liquid chromatography (1D-LC) should be accomplished in 1～10 minutes, and two-dimensional LC (2D-LC) should be accomplished in 10～100 minutes. Although it aimed at peptides, the suggestion could be also used as an important reference criterion of protein separation. Based on discontinuous migration of proteins on liquid-solid interface and new column technology of chromatographic cake, one minute-liquid chromatography (OMLC) of intact proteins which has been a dream of separation scientists for a long time is firstly established in the thesis. Compared with conventional fast separation only involving the process of protein separation itself, the OMLC includes the total time of separation and column re-generation. The OMLC was tested with reversed-phase liquid chromatography (RPLC), ion-exchange chromatography (IEC), hydrophobic-interaction chromatography (HIC) using standard proteins under the condition that in comparison with their corresponding to conventional LC, the. resolution of intact proteins in this thesis does not significantly lose. Besides high speed and high resolving power, the technology has the advantages of high loading capacity.The thesis includes the following six sections:1 IntroductionThe significance of fast separation by LC was briefly introduced. One-dimensional methods of rapid separation of intact proteins including non-porous stationary phase, monolithic stationary phase, perfusion chromatography, high temperature fast chromatography and chromatographic cake were simply introduced and reviewed. Both conventional two-dimensional liquid chromatography (2D-LC) and two-dimensional liquid chromatography using only a single column (2D-LC-1C) which exhibits excellent resolution in two modes were also introduced and reviewed.2 Separation of intact proteins with porous reversed-phase packings with high speed, high resolution and high sample loading Ultrashort chromatographic cake (1 mm×10 mm I.D.,78μL) packed with 3μm porous C18 reversed-phase packings from lateral hole of the cake was employed for the separation of seven standard proteins. The resolution for intact proteins and small solutes between such a small packed bed volume of chromatographic cake and a large pack bed volume of column (50 mm×4.6 mm I.D.,830μL) were firstly compared. Continuous migration of the small molecules in the packed bed of column and cake resulted in that the resolution of small solutes was extremely poorer with the cake than the column, but due to the existence of "adhesive energy" of intact proteins on the liquid-solid interface the discontinuous migration of intact proteins resulted in almost the comparable resolution between the cake and the column. However, the cake having better hydrodynamic property than column showed moderate back pressure at high flow rate up to 10 mL/min. Under an optimized condition, the mixture of seven proteins was completely separated in 30 s. Five consecutive separations within 5 minutes were carried out with the cake also, indicating one minute-liquid chromatography (OMLC) of intact proteins can be accomplished when the molecular interactions between protein and stationary phase is non-selective force. The OMLC has been a dream of separation scientists for a long time now becomes realism. The mixture of seven proteins was well separated within 1 minute under the sample loading of 1 mg and thus the separation of intact proteins in this circumstance also achieves the "three high" purpose of high speed, high resolution and high sample loading. The method is expected to be used in clinically routine analysis.3. Separation of intact proteins using porous weak cation-exchange packings with "three high" purposeThe comparison of separations of intact proteins with two commercial weak cation-exchange packings, TSKgel CM-5PW and PolyCAT A, was carried out. It was found that PolyCAT A (particle size,3μm; pore size,150 nm) was more suitable for achieving the OMLC. The resolution of the PolyCAT A cake (1 mm×10 mm I.D.) was comparable with the PolyCAT A column (50 mm×4.6 mm I.D.). However, the cake showed better hydrodynamic property than the column. The complete separation of 4 standard proteins was achieved in 18 s under an optimized condition with the cake. The speed and resolution is better than non-porous stationary phase and monolithic stationary phase reported in literatures. Ten consecutive separations within 10 minutes were carried out using the cake, really accomplishing the OMLC, when the molecular interaction between protein and stationary phase is electrostatic force. Also, fast separation was achieved in 1 minute under the loading of 1 mg mixture of 4 proteins.4. Fast separation of protein with non-porous HIC packingsBoth the chromatographic cake (10 mm×10 mm I.D.) and column (35 mm×4.6 mm I.D.) packed with 2.5μm non-porous HIC packings were employed for the separation of standard proteins. Under optimized conditions, the mixture of seven proteins could be separated within 3.5 min with the column, and a comparable resolution was obtained in 2.1 min with the cake. The cake, which can be used at higher flow rate, is more suitable for fast separation due to its better hydrodynamic property. Although the result is more than one minute, it is closed (2-3.5 min) to the OMLC.5. Separation of intact proteins with "three high" purpose using non-porous RPLC packingsA chromatographic cake (7.5 mm×10 mm I.D.) having larger diameter than its thickness is firstly employed to completely separate 1.0μg and 40μg of the mixture of seven standard proteins within 1 minute under conventional chromatographic conditions. Also,0.5 mg of the mixture was almost completely separated within 1 minute. The mixture of proteins was separated with high speed, high loading and high resolution. A new approach for rapid separation of proteins was put forward using conventional HPLC under high loading.6. Porous 2D (SAX-HIC) packings employed for the OMLC in HIC modeTaking the advantage of two dimensional packings (2D-packings) having a better selectivity than its corresponding one dimensional packings, a kind of porous two-dimensional packings of strong anion-exchange (SAX) and HIC was synthesized.The packed 2D (SAX-HIC) cake (1 mm×10 mm I.D.) was used to separate standard proteins in HIC mode. The mixture of six standard proteins could be almost completely separated and the cake was re-generated within one minute under the flow rate of 5 mL/min. Such a fast and good resolution could not be obtained with normally commercial HIC columns. This fact indicates that OMLC is validly carried out for the retention mechanism of protein dominated by hydrophobic-interaction force.