Integration of direct binding sensors with mass spectrometry for functional and structural characterization of molecules

A sensor and molecular mass technology, applied in the direction of instruments, scientific instruments, analytical materials, etc., to achieve the effect of simplifying the mass spectrometry method

Inactive Publication Date: 2007-10-17
SRU BIOSYST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previously known methods that combine detection assays with structural analysis are extremely limited in utility by the sub-femtomole quantities of bound material that can be recovered from flow cells, and by the requirement to produce detectable material in sufficient quantities. Extreme limitation of numerous sample injection / detection / elution cycles

Method used

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  • Integration of direct binding sensors with mass spectrometry for functional and structural characterization of molecules
  • Integration of direct binding sensors with mass spectrometry for functional and structural characterization of molecules
  • Integration of direct binding sensors with mass spectrometry for functional and structural characterization of molecules

Examples

Experimental program
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Effect test

Embodiment 1

[0071] Tandem BIND / MALDI-MS test

[0072] An assay was performed to show that materials bound to the colorimetric resonant reflectance optical sensor surface can be efficiently eluted and analyzed by mass spectrometry. Allow the capture antibody to be absorbed onto the colorimetric resonant reflectance optical sensor surface. The corresponding antigen is allowed to bind to the antibody during application of the sample on the colorimetric resonant reflectance optical sensor. Any material that is specifically bound to the antibody is then eluted from the surface and a small aliquot of the eluted material is subjected to mass spectrometric analysis. The eluted material was mixed with an appropriate amount of MALDI matrix and added to a MALDI plate for (TOF)MS analysis.

[0073] Figure 1 shows the protocol used to combine colorimetric resonant reflectance optical sensor technology with MALDI-type mass spectrometry. The procedure involved adsorption of antibody (0.1 mg / ml) on a ...

Embodiment 2

[0075] Matrix Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry (MS)

[0076] The colorimetric resonant reflectance optical sensor TiO sensor was pre-rinsed three times with PBS and placed at room temperature for 30 minutes, a baseline reading was taken for a few minutes and 10ul of 1mg / ml human IgG or chicken IgY was diluted into the wells already in the well. 90ul PBS to form a final antibody concentration of 100ug / mL. Proteins were placed into the wells and allowed to bind to the TiO surface for 90 minutes. Unbound protein solution was removed from the wells and the wells were rinsed three times with 200ul of PBS each.

[0077] Another baseline reading is taken for a few minutes, then 1 ul of 1 mg / ml anti-human IgG (Fab) is placed in wells that are coated with hIgG (red) or cIgY (yellow) and allowed to incubate for 60 minutes . All unbound protein solution was removed from the wells, which were rinsed three times with PBS. Monitor the stability of the bound...

Embodiment 3

[0080] Tandem BIND / MALDI-MS Test - MS Data

[0081] Figure 3A shows data from a Fab-containing control solution applied to MS prior to exposure under the sensor surface. The main peak is at 22300, and the other two peaks are characteristic peaks related to the parent molecular mass.

[0082] Figure 3B shows MALDI-MS data from the solution eluted from the sensor surface. This mass spectrum is identical to the control spectrum shown in Figure 3A.

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Abstract

The invention provides methods for the detection, quantification, identification and structural analysis of one or more molecules. Mass spectrometry (MS) is not a universal detector as all molecules do not ionize equally well leading to poor signal to quantity information. MS can be optimized to identify the specific mass of a binding component when the presence of a material is known. Colorimetric resonant reflectance optical sensors provide a universal mass detector in that nearly all biological masses give equally proportional signals. The combined methods allow selection and or detection with quantification of all masses binding to the sensor with the ability to identify specific molecules by their individual masses and structure analyses.

Description

[0001] priority [0002] This application claims the benefit of US Serial Application No. 60 / 583,560, filed June 28, 2004, which is hereby incorporated by reference in its entirety. Background of the invention [0003] Structural analysis combined with detection assays can provide complementary information on molecular function and structure. Previous work has demonstrated the utility of this combinatorial approach for applications such as ligand fishing, epitope mapping and amino acid sequencing, but only in systems based on microfluidic channel surface plasmon resonance (SPR). Where low-throughput samples are limited. See Nelson et al., BIA / MS of Epitope-Tagged Peptides Directly from E. coli Lysate: Multiplex Detection and Protein Identification at Low-Femtomole to Subfemtomole Levels. Analytical Chemistry 1999, 71:2858-2865; Nelson et al., Biosensor chip mass spectrometry: A chip -based proteomics approach. Electrophoresis 2000, 21: 1155-1163. Previously known methods th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/27G01N33/487G01N33/543
CPCG01N33/54373G01N21/27G01N33/6848G01N33/542G01N33/543
Inventor 李允中B·林C·威廉斯L·梁
Owner SRU BIOSYST
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