Mass labels

a technology of mass labels and labels, applied in the field of mass labels, can solve the problems of increasing the complexity of the mass spectrum, not always possible available synthetic standards, etc., and achieve the effect of limiting the multiplexing rate of a single isobaric mass tag set, rapid and inexpensiv

Inactive Publication Date: 2015-03-05
ELECTROPHORETICS LTD
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  • Abstract
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Benefits of technology

[0009]The inventors found that by using a common core structure, preferably based on DMPip-βAla it was possible to develop a range of products with selective labelling properties and / or additional offset masses which circumvent the need to re-design workflows or software for interpretation of quantitative mass spectrometry data. In addition, they have shown that it is possible to use the same core structure to develop isochemic tags offering the benefit of quantitation in LC-MS with direct conversion to equivalent isobaric mass tags for biomarker qualification and / or clinical assay development.
[0038]Embodiment b) above comprises the 2-dithiopyridine group and has several advantages: it shows a high selectivity to label cysteine residues, even at increased pH as often used in buffer solutions useful in proteomic investigations (eg. Triethylammonium bicarbonate TEAB) and it is not labile to exposure to water. Furthermore, this group can be re-cleaved from peptides easily if desired by treatment with any disulfide-reducing reagents.
[0047]Such reactive mass labels have improved aqueous solubility and stability. The majority of mass tags carry a succinimide ester group to allow efficient labelling of amine functions on peptides and proteins. Whilst the succinimide labelling reaction is rapid and can be driven to completion with relatively low molar excess, it is highly sensitive to hydrolysis of the succinimide ester. In certain applications such as labelling of cell surfaces it is necessary to employ a predominantly aqueous environment and the use of a standard succinimide ester is not possible. Improvements to succinimide esters have been made with sulfo-N-hydroxysuccinimide ester (sulfo-NHS) showing a greater resistance to hydrolysis than the non-sulfonated parent. An additional benefit of the sulfo-NHS type of group is that it renders the tandem mass tag highly polar and prevents uptake of tags into the cell through the intact cell membrane. Consequently the sulfo-NHS derivatives of TMT are specifically able to label extracellular proteins. However, removal of free sulfo-NHS during mass tag manufacture can be problematic. To circumvent this, the present inventors discovered that a sulfo-tetrafluorophenyl moiety could be used.

Problems solved by technology

However, these methods have traditionally required an available synthetic standard which is not always possible.
Whilst isochemic tags allow a degree of improvement in the reproducibility of proteomic studies, this is achieved at the cost of increasing the complexity of the mass spectrum.

Method used

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example 1

Synthesis of dimethylpiperidine-βalanine-dithiopyridine: A Cysteine-Reactive Mass Label

[0310]It is a convenient feature of the DMPip-βALA-OSu structure that alternate reactivities can be readily created by reaction with the succinimide ester reactive group. Thus, using the known TMT (Tandem Mass Tag or mass label) structure previously disclosed in WO2007 / 012849 as a starting point and commonly available building blocks, a reaction scheme was designed which requires only one more reaction step to convert the amino-reactive compound into the respective cysteine-reactive compound. We first synthesised the dithiopyridine modification reagent from commercially available compounds in a single reaction with good yield. This reagent was then allowed to react with DMPip-βALA-OSu to generate the DMPip-βALA-DTP reagent (see FIG. 2). Analysis by HPLC, MS and MS / MS revealed the correct identity and a purity >90%.

example 2

Labelling of Synthetic Peptides Using DMPip-βALA-DTP

[0311]The quality controlled DMPip-βALA-DTP reagent was then applied to develop a labelling protocol. An individual cysteine-containing peptide (H-Vat-Ala-Thr-Val-Cys-Leu-Pro-Arg-OH) was chosen to allow for an easy monitoring of the reaction progress by HPLC with UV-readout. A protocol was developed that yields an essentially complete labelling. 50 μg BSA were dissolved in 100 μL TEAB (100 mM, pH 8.5) including 0.1% SDS. After reduction with 1 mM of tris[2-carboxyethyl]phosphine * HCl for 1 h at 55° C., the Cys residue was labelled with 5 mM DMPip-βALA-DTP (provided as 200 mM stock solution in methanol). Purification of the reaction mixture using RP and SCX cartridges obtained modified peptide in a highly purified form. FIG. 3 shows HPLC monitoring of the individual species in the labelling reaction.

example 3

Synthesis of Mass Labels for Steroid Analysis

[0312]The analysis of hormones is typically performed by radioimmunoassay and colorimetric or chemiluminescent enzyme immunoassay. However, current immunoassays are disadvantageous since they are limited to quantitative detection of only one steroid hormone per assay, may lack specificity, and as a result of such cross-reactivity can show up to 15 fold variance in the quantitative results of the same sample when using kits from different manufacturers.

[0313]An alternate approach for measurement of steroids is gas chromatography-mass spectrometry (GC-MS). GC-MS is both sensitive and specific, but requires tedious and time-consuming sample preparation. Liquid chromatography-MS (LC-MS) and liquid chromatography-tandem MS are equally specific and offer simpler approaches to sample preparation often without such complex sample derivatization steps. Recently, a number of LC-MS-based methods using different ion sources have been reported for the...

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Abstract

A reactive mass label for labelling a biological molecule for detection by mass spectrometry includes the following structure: X-L-M-S-Re, wherein X is a mass marker moiety, L is a cleavable linker, M is a mass normalization moiety, and S is a mass series modifying group including the following group:wherein J is C═O, K is NH, and n is 2 or J and K are both CH2 and n is 1, m is at least 1; and Re is a reactive functionality for attaching the reactive mass label to a biological molecule.

Description

CROSS-REFERENCE TO RELATED APPLICATION(S)[0001]This application is a divisional of U.S. application Ser. No. 13 / 497,875 filed Jul. 23, 2012, which is a U.S. National Stage Application filed under 35 U.S.C. §371 and claims priority to International Application No. PCT / EP2010 / 063191, filed Sep. 8, 2010, which application claims priority to Great Britain Application No. 0916881.6, filed on Sep. 25, 2009, the disclosures of which are incorporated by reference herein in their entirety.FIELD OF THE INVENTION[0002]This invention relates to compounds for labelling analytes, particularly biomolecules such as proteins. This invention also relates to methods of analysis by mass spectrometry, using specific mass labels.BACKGROUND TO THE INVENTION[0003]The field of human medicine has been dependent on the ability to detect changes caused by or in response to disease. Such changes provide means of diagnosis and offer insights to the targets for therapeutic compounds such as vaccines and medicines...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/74C07D211/14G01N33/58C07D401/12
CPCG01N33/743C07D401/12G01N2458/15G01N33/58G01N2560/00C07D211/14C07B59/002Y10T436/200833Y10T436/24Y10T436/196666
Inventor BAUMANN, CHRISTIANKIENLE, STEFANKUHN, KARSTENLEGNER, HARALD
Owner ELECTROPHORETICS LTD
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