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

Inventors

Lim, Mark J. • Yagnik, Gargey • Rothschild, Kenneth J.

Assignees

Ambergen Inc

Abstract

The field of this invention relates to immunohistochemistry (IHC) and in situ hybridization (ISH) for the targeted detection and mapping of biomolecules (e.g., proteins and miRNAs) in tissues or cells for example, for research use and for clinical use such by pathologists (e.g., biomarker analyses of a resected tumor or tumor biopsy). In particular, the use of mass spectrometric imaging (MSI) as a mode to detect and map the biomolecules in tissues or cells for example. More specifically, the field of this invention relates to photocleavable mass-tag reagents which are attached to probes such as antibodies and nucleic acids and used to achieve multiplex immunohistochemistry and in situ hybridization, with MSI as the mode of detection/readout. Probe types other than antibodies and nucleic acids are also covered in the field of invention, including but not limited to carbohydrate-binding proteins (e.g., lectins), receptors and ligands. Finally, the field of the invention also encompasses multi-omic MSI procedures, where MSI of photocleavable mass-tag probes is combined with other modes of MSI, such as direct label-free MSI of endogenous biomolecules from the biospecimen (e.g., tissue), whereby said biomolecules can be intact or digested (e.g., chemically digested or by enzyme).

Core Innovation

The invention relates to photocleavable peptide mass-tags conjugated to biomolecular probes to enable multiplexed, targeted mass spectrometric imaging of biomarkers in tissue and cells. The probes include antibodies, nucleic acids or aptamers, and other probes such as lectins, receptors, and ligands, where each probe is conjugated to a unique photocleavable mass-tag for identifying bound biomarker targets.

The method provides a multiplex method for co-detecting 3 or more different biomarkers in a tissue sample by contacting the tissue sample with 3 or more different probes in a mixture to create a probed tissue sample, where each probe is conjugated to a unique photocleavable mass-tag and at least 3 probes bind to different biomarkers in the tissue sample. After probe binding, the mass-tags are photocleaved with an exogenous source of UV light, and the resulting mass-tag species are detected by mass spectrometric imaging.

The unique mass-tags or fragments thereof are detected as molecular ions, allowing co-detection of three or more different biomarkers in a single tissue sample. The disclosed system also supports multi-omic imaging combinations, including combining targeted PC-MT MSI with label-free untargeted MSI on the same tissue section, and performing sequential double MSI workflows.

Claims Coverage

The provided independent claim is clm-00001, and the inventive-feature coverage centers on three main inventive features.

The core inventive coverage is the use of multiple biomarker-binding probes each conjugated to a unique photocleavable mass-tag, followed by UV photocleavage and mass spectrometric imaging detection of the unique mass-tags or fragments as molecular ions.

Stated Advantages

Documented Applications