Novel photocleavable mass-tags for multiplexed mass spectrometric imaging of tissues using biomolecular probes

Abstract

A multiplex method for co-detecting a plurality of different types of biomarkers in a tissue sample on a single slide comprises: (a) providing a tissue sample on a single slide; (b) performing antigen

Claims

1. A multiplex method for co-detecting a plurality of different types of biomarkers in atissue sample on a single slide, said method comprising:a) providing a tissue sample on a single slide;b) performing antigen retrieval on said tissue sample;c) contacting said tissue sample with a plurality of different antibodies to effect binding of the antibodies to the tissue sample, each of said antibodies reactive with a different biomarker, and each of said antibodies conjugated to a unique photocleavable mass-tag;d) illuminating said photocleavable mass-tags with an exogenous source of UV light so as to photocleave at least a portion of said mass-tags prior to step d); ande) detecting, using mass spectrometric imaging, said mass-tags, or fragments thereof, as molecular ions.

2. The method of claim 1, further comprising performing, after step a) but before step b), direct mass spectrometric imaging on said tissue sample.

3. The method of claim 1, wherein said plurality of different antibodies are in a mixture and said tissue sample in step c) is contacted with said mixture.

4. The method of claim 1, wherein said mass-tags are non-rare-earth-metal mass-tags.

5. The method of claim 1, wherein said mass-tags comprise a plurality of amino acids.

6. The method of claim 1, wherein said tissue sample was fresh frozen and thin-sectioned prior to being mounted on said single slide.

7. The method of claim 1, wherein said slide comprises gold.

8. The method of claim 7, wherein said slide is a glass slide with a gold layer.

9. The method of claim 1, wherein said tissue sample was formalin-fixed and paraffin embedded and thin-sectioned prior to being mounted on said single slide.

10. The method of claim 9, wherein the tissue sample is subjected to a treatment prior to the steps of contacting the sample with antibody, said treatment comprising deparaffinization.

11. The method of claim 10, wherein said deparaffinization is performed with xylene.

12. The method of claim 10, wherein the tissue sample is further subjected to a treatment, said treatment comprising rehydration.

13. The method of claim 12, wherein said rehydration is performed with a series of ethanol / water mixtures and aqueous saline buffers.

14. The method of claim 1, wherein said antigen retrieval is performed by heating in citrate buffer, pH 6.

15. The method of claim 1, wherein said antigen retrieval is performed with the use of formic acid.

16. The method of claim 1, wherein a matrix compound is applied to said mass-tags after step d) and before step e).

17. The method of claim 16, wherein said matrix compound is selected from the group consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-Dihydroxybenzoic acid (DHB) and 3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid).

18. The method of claim 16, wherein said matrix compound is applied by sublimation.

19. The method of claim 16, wherein the tissue sample is subjected to a treatment after the steps of contacting the sample with a matrix compound, said treatment comprising matrix recrystallization.

20. The method of claim 1, wherein said photocleavable mass-tags conjugated to said antibody probes comprise a core structure which is conjugated to a mass unit, said antibody probes having the following general structure: Mass Unit - Core Structure -Antibody.5 21. The method of claim 1, wherein said tissue is from a tumor.

22. The method of claim 21, wherein said tumor is a breast tumor.

23. The method of claim 1, wherein at least one of said plurality of different antibodies10 comprises a fluorescent moiety in addition to said mass-tag.

24. The method of claim 1, wherein the exogenous source of UV is an LED UV source.Application No: GB2501307.9Examiner: Dr Radhe ShyatnClaims searched: 1-24Date of search: 17 February 2025Patents Act 1977: Search Report under Section 17Documents considered to be relevant:Category Relevant to claims Identity of document and passage or figure of particular relevance X 1-24 Rapid communications in mass spectrometry : RCM, vol. 28, no. 1, 2014, Lim Mark J et al., Correlated matrix-assisted laser desorption / ionization mass spectrometry and fluorescent imaging of photocleavable peptide-coded random bead-arrays., pages 49-62. See whole document, especially figure 2 and the Discussion at pages 60 to 61 X 1-24 US 2019 / 0004038 Al (LIM et al.) See whole document, especially para 51, 76 and 174 A - Nucleic Acids Research, vol. 27, no. 23, 1999, OLEJNIK J ET AL, Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS., pages 4626-4631. See whole document, particularly figure 1 and last para of the introduction A Tetrahedron, vol. 72, no. 36, 2016, Kang Nana et al., Design and synthesis of new mass tags for matrix-free laser desorption ionization mass spectrometry (LDLMS) based on 6,ll-dihydrothiochromeno[4,3-b]indole, pages 5612-5619. See whole document, particularly figure 2 A - WO 2011 / 006075 Al (SOMALOGIC INC) See whole document, particularly para [00177]-[00178] A US 2016 / 0139143 Al (HONG et al.) See para [0377], [0401] and particularly example 21 and para [0469] A - WO 2020 / 176788 Al (10X GENOMICS INC) See page 72 under immunohistochemistry and immunofluorescence section, lines 23-27Categories:v Au Document indicating lack of novelty or inventive step A Document indicating technological background and or state of the art. Y Document indicating lack of inventive step if combined with one or more other documents of same category. P Document published on or after the declared priority date but before the filing date of this invention. & Member of the same patent family E Patent document published on or after, but with priority date earlier than, the filing date of this application.Field of Search:International Classification:Subclass Subgroup Valid From GOIN 0033 / 574 01 / 01 / 2006 GOIN 0033 / 50 01 / 01 / 2006 GOIN 0033 / 533 01 / 01 / 2006 GOIN 0033 / 68 01 / 01 / 2006 H01J 0049 / 00 01 / 01 / 2006 H01J 0049 / 16 01 / 01 / 2006

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