Use of 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:5 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;10 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 e), wherein after photocleavage the mass tag has a terminal amine group; 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 and20 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.

256. 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.

308. The method of claim 7, wherein said slide is a glass slide with a gold layer.22 07 259. 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 the5 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,10 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.20 16. The method of claim 1, wherein a matrix compound is applied to said mass-tags after stepd) 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)25 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 the30 steps of contacting the sample with a matrix compound, said treatment comprising matrix recrystallization.22 07 2520. The method of claim 1, wherein said photocieavable 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.22 07 25

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