Probe for mass spectrometry

a mass spectrometry and probe technology, applied in the field of probes for mass spectrometry, can solve the problems of disfavorable radiolabels, limited current methods for analysing protein microarrays, and limitations in both methods, and achieve the effect of more accurate measuremen

Inactive Publication Date: 2005-09-08
KOOPMAN JENS OLIVER +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0067] In contrast to the prior art in which matrix and analyte are co crystalised in an aqueous solvent, the Applicant uses two distinct steps in which first the protein is deposited in an aqueous solvent and then the energy absorbing molecules are deposited such that they crystallise out from the non aqueous solvent on the probe. This has the advantage that the protein is deposited in its biological form However, u...

Problems solved by technology

However, both methods suffer limitations due to their bias towards highly expressed proteins and the destructive method of separation.
The current methods of analysing protein microarrays are therefore restricted by the availability of appropriate labelled ligands.
However, for drug-like small molecules, which often have molecular weights of less than 1000 Da, neither radio- or fluorescent labels are desirable; radiolabels are disfavoured for health and safety reasons, whilst the introduction of a fluorophore into the small molecule could significantly perturb the structure activity profile in an unpredictable manner.
However, the development of a MALDI MS-compatible protein microarray is complex since existing methods for forming protein microarrays do not transfer readily onto to a MALDI target.
There are a number of reasons why this is the case, inter alia the specialised nature of the probe surfaces and the potential for salts present in reaction buffers to interfere with the detection method.
As a consequence it is often observed that individual crystals contain ...

Method used

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  • Probe for mass spectrometry
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Examples

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example 1

[0121] Affinity capture of a variety of tagged proteins can be demonstrated using for example PEG-PLL-biotin or PEG-PLL-Bleomycin B6 as the protein capturing moieties.

[0122]FIGS. 2, a and b show the mass spectra acquired from a protein microarray demonstrating respectively the capture of 1500 and 15 femtogram of biotin tagged insulin. The biotin tagged insulin was arrayed onto an affinity capture surface on a gold coated microscope glass slide in a 3 nanoliter volume using 300 micrometer pins (Q-Array, Genetix, New Milton, UK). The gold coated PEG-PLL-Biotin Neutravidin surface, was washed three times with 1 mM Tris-HCl pH 7.5, dried under a stream of nitrogen and overlaid with 3 nanolitre of α-cyano-4-hydroxy-cinnamic acid dissolved in 99% acetone v / v, 1% glycerol resulting in a spot with an radius of approximately 200 micrometer. The probe was analysed with a mass spectrometer MALDI TOF mass spectrometer. Several biotin tagged insulin peaks are visible due to the different degree...

example 2

[0124] A PLL-PEG-biotin neutravidin surface on a MALDI target is overlaid with 500 nanoliters of a biotinylated protein mixture derived from an E. coli lysate expressing a human recombinant protein in conjunction with a sequence tag in this case Biotin carboxyl carrier protein (BCCP) from E. coli. The protein was captured for a period of 2 hours on the surface, washed twice with washing buffer followed by two washes with desalting buffer, and overlaid with 300 nanoliters of an energy absorbing matrix, namely saturated α-cyano-4-hydroxycinnamic acid in acetone. The mass spectrum acquired in linear mode using the delayed extraction technique at low laser power is illustrated in FIG. 5. The advantage of this method is that the sample can be applied as a complex mixture of proteins and after washing only the molecules of interest remain. Secondly the analyte is captured in a spatially defined position before it is released from the affinity capture surface by the addition of matrix.

4....

example 3

[0125]FIG. 6 shows the peptide fingerprint analysis of Glutathione-S-transferase-Biotin Carboxyl Carrier Protein (GST-BCCP). A bacterial crude lysate containing the fusion protein and bacterial proteins was placed on the MALDI target previously coated with PEG-PLL-biotin and neutravidin. The BCCP fusion partner of GST contained a biotinylation consensus sequence such that it becomes biotinylated when expressed in E. coli. allowing the fusion protein to bind specifically to the PEG-PLL-biotin neutravidin surface, whilst allowing the bacterial proteins to be washed away with buffer. For identification purpose the surface captured protein was digested by overlaying it with trypsin and analysed by MALDI MS. A protein fingerprint analysis revealed 12 peptides belonged to GST from Schistosoma mansoni, 4 peptides belonging to Neutravidin and 3 to trypsin (see table 1), but no bacterial protein was identified using the remaining unmatched peptides. This experiment demonstrates that PEG-PLL-...

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Abstract

The present invention relates to a probe for the analysis of one or more analytes, particularly proteins or compounds capable of binding or otherwise interacting therewith, by laser desorption/ionisation mass spectrometry, more particularly MALDI MS. It also relates to a protein microarray, a method of producing a protein microarray and a method of analysing a protein microarray. The probe comprises a support having an electroconductive target surface thereon characterized in that the target surface comprises a micro array having a plurality of discrete target areas presenting one or more analyte capture moieties. Each discrete target area has an area of less than 1000 μm2, more preferably still less than 500 μm2, and more preferably still less than 100 μm2.

Description

[0001] The present invention relates to a probe for the analysis of one or more analytes, particularly proteins or compounds capable of binding or otherwise interacting therewith, by laser desorption / ionisation mass spectrometry, more particularly MALDI MS; It also relates to a protein microarray, a method of producing a protein microarray and a method of analysing a protein microarray. [0002] Such a mass spectrometry probe, upon which a microarray has been fabricated, enables interrogation of protein—small molecule interactions in a label-free manner by desorption and ionisation of analytes (e.g. protein, drug or drug candidate, carbohydrate, DNA, RNA or other test molecule). The probe and methods are particularly useful in the drug discovery process, for example in hit series evaluation, lead optimisation, predictive toxicogenomics and metabolite profiling. [0003] Analysis of disease processes and drug effects have traditionally focussed on genomics, whereas proteomics, the study ...

Claims

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Application Information

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IPC IPC(8): G01N27/64A61K47/48C07K14/00G01N27/62G01N33/483G01N33/53G01N33/94G01N37/00H01J49/04H01J49/16
CPCA61K47/48407B82Y30/00C07K14/00Y10T436/24G01N2415/00H01J49/0418G01N33/9446A61K47/6809
Inventor KOOPMAN, JENS-OLIVERBLACKBURN, JONATHAN M.
Owner KOOPMAN JENS OLIVER
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