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(12) Patent Application Publication (10) Pub. No.: US 2005/0269267 A1 Patton Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2005/0269267 A1 Patton Et Al US 2005O269267A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0269267 A1 Patton et al. (43) Pub. Date: Dec. 8, 2005 (54) SEPARATIONS PLATFORM BASED UPON Publication Classification ELECTROOSMOSIS-DRIVEN PLANAR CHROMATOGRAPHY (51) Int. Cl. ................................................. B01D 15/08 (52) U.S. Cl. ......................... 210/656; 210/748; 204/450; (75) Inventors: Wayne F. Patton, Newton, MA (US); 436/161 Mack J. Schermer, Belmont, MA (US) Correspondence Address: (57) ABSTRACT WILMER CUTLER PICKERING HALE AND DORR LLP 60 STATE STREET The present invention describes a System and method for BOSTON, MA 02109 (US) Separation of proteins, peptides and glycans by one-dimen Sional or two-dimensional electroosmosis-driven planar (73) Assignee: PerkinElmer LAS, Inc., Boston, MA chromatography. Separation is performed using amphiphilic polymeric membranes, amphiphilic thin-layer chromatogra (21) Appl. No.: 11/084,501 phy plates or other planar amphiphilic Surfaces as the (22) Filed: Mar. 18, 2005 Stationary phase with a combination of organic and/or aque ouS buffers as the mobile phase. Systematic Selection of Related U.S. Application Data Stationary phase Supports, mobile phase buffers and operat ing conditions allow for the adaptation of the invention to a (60) Provisional application No. 60/521,250, filed on Mar. broad range of applications in proteomics, mass spectrom 19, 2004. etry, drug discovery and the pharmaceutical Sciences. a s Patent Application Publication Dec. 8, 2005 Sheet 1 of 13 US 2005/0269267 A1 Patent Application Publication Dec. 8, 2005 Sheet 2 of 13 US 2005/0269267 A1 s Patent Application Publication Dec. 8, 2005 Sheet 3 of 13 US 2005/0269267 A1 Patent Application Publication Dec. 8, 2005 Sheet 4 of 13 US 2005/0269267 A1 s Patent Application Publication Dec. 8, 2005 Sheet 5 of 13 US 2005/0269267 A1 Patent Application Publication Dec. 8, 2005 Sheet 6 of 13 US 2005/0269267 A1 Patent Application Publication Dec. 8, 2005 Sheet 7 of 13 US 2005/0269267 A1 Patent Application Publication Dec. 8, 2005 Sheet 8 of 13 US 2005/0269267 A1 Patent Application Publication Dec. 8, 2005 Sheet 9 of 13 US 2005/0269267 A1 Patent Application Publication Dec. 8, 2005 Sheet 10 of 13 US 2005/0269267 A1 Patent Application Publication Dec. 8, 2005 Sheet 11 of 13 US 2005/0269267 A1 Patent Application Publication Dec. 8, 2005 Sheet 12 of 13 US 2005/0269267 A1 Patent Application Publication Dec. 8, 2005 Sheet 13 of 13 US 2005/0269267 A1 US 2005/0269267 A1 Dec. 8, 2005 SEPARATIONS PLATFORM BASED UPON watery ink Spots on a wobbly, Sagging, gelatinous-like slab. ELECTROOSMOSIS-DRIVEN PLANAR Other limitations include difficulty in automating the Sepa CHROMATOGRAPHY ration process, low throughput of Samples, and difficulty in detecting low abundance, extremely basic, very hydropho CROSS-REFERENCE TO RELATED bic, very high molecular weight or very low molecular APPLICATIONS weight proteins. While detection of proteins directly in gels with labeled antibodies or lectins has been accomplished, 0001. The application claims benefit of U.S. provisional the approach is not generally applicable to every antigen and patent application No. 60/521,250, filed Mar. 19, 2004. is relatively insensitive. Consequently, proteins are usually electrophoretically transferred to polymeric membranes FIELD OF THE INVENTION before Specific targets are identified. The polyacrylamide gel 0002 The present invention generally relates to the sepa also poses difficulties in the identification of proteins by ration of proteins, peptides and glycans using electrooSmo microchemical characterization techniques, Such as mass Sis-driven planar chromatography. The present invention Spectrometry, Since the gels must be macerated and rinsed, also relates to Systems and methods for Separating biomol the proteins must be incubated with proteolytic enzymes, ecules using planar electrochromatography. and peptides must be Selectively retrieved and concentrated using a reverse-phase column prior to identification. BACKGROUND OF INVENTION 0006 Integral membrane proteins play an important role 0003. The human proteome is known to contain approxi in Signal transduction and are thus primary drug targets mately 30,000 different genes. But, due to post-translational pursued by the pharmaceutical industry. The proteins typi modifications and differential mRNA splicing, the total cally contain one or more hydrophobic, transmembrane number of distinct proteins is most likely to be close to one domains that intermingle with the hydrophobic portion of million. The level of complexity, coupled with the relative lipid bilayer membranes. The 2DGE technique is poorly abundances of different proteins, presents unique challenges Suited for the fractionation of hydrophobic proteins, particu in terms of Separations technologies. Analytical methods for larly proteins containing two or more alpha-helical trans the Simultaneous quantitative analysis of the abundances, membrane domains, because the technique is based upon locations, modifications, temporal changes and interactions aqueous buffers and hydrophilic polymers. of thousands of proteins are important to proteomics. Two 0007 Two-dimensional liquid chromatography-tandem dimensional or even multi-dimensional protein separations, mass spectrometry (2D LC/MS/MS) has been used as an based upon different physicochemical properties of the alternative analytical approach for protein Separation. In 2D constituent proteins, are favored over Single dimension LC/MS/MS, a proteolytic digest of a complex protein Separations in proteomics due to the increased resolution Sample is loaded onto a microcapillary column that is afforded by the additional dimensions of fractionation. Two packed with two independent chromatography phases, a dimensional Separation Systems can be categorized by the Strong cation exchanger and a reverse-phase material. Pep type of interface between the dimensions. In "heart-cutting tides are iteratively eluted directly into a tandem mass methods a region of interest is Selected from the first Spectrometer and the Spectra generated are correlated to dimension and the Selected region is Subjected to Second theoretical mass spectra obtained from protein or DNA dimension Separation. Systems that Subject the entire first databases. This peptide-based approach to proteomics gen dimension to a Second dimension Separation, or alternatively erates large number of peptides from a specimen that Sample the effluent from the first dimension at regular exceeds the analytical capacity of the LC-MS system. Con intervals and fixed Volumes for Subsequent fractionation in Sequently, Strategies have been developed that retrieve a the Second dimension, are referred to as “comprehensive” Small percentage (3-5%) of the peptides from a complex methods. digest, Such as tryptic peptides containing only cysteine 0004. The principal protein separation technology used residues or only histidine residues. The remaining 95-98% today is high-resolution two-dimensional gel electrophoresis of the peptides are discarded, thus prohibiting a comprehen (2DGE). High resolution 2DGE involves the separation of Sive analysis of the Sample. Additionally, Such procedures proteins in the first dimension according to their charge by are unable to distinguish among the various protein isoforms isoelectric focusing and in the Second dimension according exhibited in a proteome that arise from differential mRNA to their relative mobility by sodium dodecyl sulfate poly Splicing and post-translational modification due to a com acrylamide gel electrophoresis. The technique is capable of bination of poor Sequence coverage and the Sequence Scram Simultaneously resolving thousands of polypeptides as a bling arising from the fragmentation proceSS itself. constellation pattern of Spots, and is used for the global 0008 Another technique applied to the analysis of pep analysis of metabolic processes Such as protein Synthesis, tides and proteins is capillary electrochromatography glycolysis, gluconeogenesis, nucleotide biosynthesis, amino (CEC), but its use has been limited to 1-D capillary sepa acid biosynthesis, lipid metabolism and StreSS response. It is rations of model analytes. CEC is a hybrid Separation also used for the analysis of Signal transduction pathways, to technique that couples capillary Zone electrophoresis (CZE) detect global changes in Signaling events, as well as to with high-performance liquid chromatography (HPLC). In differentiate between changes in protein expression and CEC, both chromatographic and electrophoretic processes degradation from changes arising through post-translational determine the magnitude of the Overall migration rates of the modification. analytes. Unlike HPLC, where the dominant force is hydrau 0005 Polyacrylamide gels are mechanically fragile, sus lic flow, the driving force in CEC is electroosmotic flow. ceptible to Stretching and breaking during handling. Analy When a high Voltage is applied, positive ions accumulate in sis using 2DGE produces a random pattern of Smudged, the electric double layer of the particles in the column US 2005/0269267 A1 Dec. 8, 2005 packing and move towards the cathode, dragging the liquid Steps of providing a Sample comprising one or more bio phase with them. The separation mechanism in CEC is based molecules, loading the Sample on a planar Stationary phase, upon both kinetic processes (electrokinetic migration) and wherein the Stationary phase is amphiphilic, contacting the thermodynamic
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