Narrow bore layer open tube capillary column and uses thereof

Abstract

A polymer-based PLOT column prepared by in situ copolymerization of a functional monomer, which usually contains the retentive chemistries, and a crosslinking monomer, which enhances the strength of the polymer matrix, is disclosed. Styrenic based monomers such as styrene and divinylbenzene or meth / acrylic based monomers such as butyl or stearyl methacrylate and ethylene glycol dimethacrylate, are preferred. Columns of the invention can be prepared in a robust fashion with a very narrow i.d., e.g., 5-15 μm. Thus, they are suitable for commercial use in ultratrace LC / MS proteomic analysis. Columns according to the invention are characterized by high resolving power, high column-to-column reproducibility and relatively high loading capacity. When these columns are coupled on-line with, e.g., ESI-MS detection, the resulting systems provide high sensitivity for analysis of complex proteomic samples, even down to the low attomole to sub-attomole level.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60 / 815,314, filed on Jun. 21, 2006, the disclosure of which is incorporated by reference herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]Part of the work leading to this invention was carried out with United States Government support provided under a grant from the National Institutes of Health, Grant No. GM-15847. Therefore, the U.S. Government has certain rights in this invention.BACKGROUND OF THE INVENTION[0003]Electrospray ionization-mass spectrometry (ESI-MS) has become a routine tool in proteomic studies, primarily due to its high sensitivity, broad dynamic range, and versatility for online coupling with capillary high performance liquid chromatography (HPLC)1-3. High-resolution separation prior to MS detection allows complex mixtures to be characterized by extending both the dynamic range and detection...

AI Technical Summary

Benefits of technology

[0007]A new polymer-based PLOT column prepared by in situ copolymerization of a functional monomer, which usually contains the retentive chemistries, and a crosslinking monomer, which enhances the strength of the polymer matrix, is disclosed herein. For example, styrenic based monomers such as styrene and divinylbenzene or meth / acrylic based monomers such as butyl or stearyl methacrylate and ethylene glycol dimethacrylate, are employed. Columns of the invention can be prepared in a robust fashion with a very narrow i.d., e.g., 5-15 μm. Thus, they are suitable for commercial use in ultratrace LC / MS proteomic analysis. Columns according to the invention are characterized by high resolving power, high column-to-column reproducibility and relatively high loading capacity. When coupled on-line with, e.g., ESI-MS detection, these columns, in systems according to the invention, provide high sensitivity for analysis of complex proteomic samples, even down to the low attomole to sub-attomole level. The power of methods using columns of the invention is demonstrated in particular by coupling such columns to the new mass spectrometers, such as the hybrid linear ion-trap / FT mass spectrometer (LTQ / FT-MS, ThermoElectron, San Jose, Calif.), for bioanalyses. The high resolution and sensitivity of these columns opens up major possibilities for the diagnosis of biopsy samples as well as the determination of specific biomarkers that can provide molecular phenotyping of individual samples. Such developments are of clear clinical importance and therapeutic significance in that tissue samples of a highly limited quantity can be successfully analyzed for proteomic content using the columns and methods of the invention. Also, columns according to the invention can be online coupled to other sensitive detectors such as fluorescence, electro / chemiluminence or nuclear magnetic resonance (NMR) for, e.g., detection of trace chemical or biological agents in chemical or biological defense applications.

Problems solved by technology

However, even with such columns, LC-MS analysis of very low quantity samples (e.g., cells from small tissue samples obtained using laser capture microdissection4) can still be problematic.

10 nL / min 6. However, in all these cases, preparation of the monolithic columns was difficult, in part due to the increased surface area to col

However, such columns provided low retention and low sample loading capacity even for small molecules, let alone for complex biological samples.

Although efforts have been made in the last 20 years to prepare PLOT capillary LC columns20-23, success has been limited due in part to the following: 1) lack of a sensitive, universal, small dead volume detector24; 2) lack of ability to generate effective gradient elution at very low flow rates; and 3) difficulties in the preparation of capillary columns with a uniform stationary layer reproducibly.

The remaining problem is to prepare and implement high efficiency LC PLOT columns, a major challenge being to cast a suitably uniform porous layer on the column to provide sufficient retention and sample loading capacity.

However, these and other attempts32-36 have not been sufficiently successful to permit commercial level development of PLOT capillary LC columns and their use, e.g., in ESI-MS.

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