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 that are attached to probes, including antibodies, nucleic acids, and lectins/receptors/ligands, to enable read out by mass spectrometric imaging. The photocleavable peptide mass-tags are used for multiplex immunohistochemistry and in situ hybridization, with detection performed by mass spectrometric imaging read out of the photocleaved peptide mass-tags.

The problem addressed is the need for multiplex detection in situ, enabling visualization of multiple biomarkers on a single slide while maintaining probe-based targeting. The described approach combines photocleavable mass-tags with mass spectrometric imaging to provide multiplex immunohistochemistry and in situ hybridization read out.

The workflow is described as compatible with matrix-assisted MALDI-MSI and also with DESI-MSI, including matrix sublimation and matrix recrystallization, and it further enables optional fluorescence co-labeling as part of the probe-based multiplex detection context. The invention also includes a general chemical structure for a photocleavable mass-tag linker system in which a first linker unit comprises at least 1 amine, the amine nitrogen is covalently bonded to an Fmoc or Boc protecting group, and a second linker unit is included.

Claims Coverage

The independent claim defines a composition by a general chemical structure containing two linker units and a nitrogen-protecting group. The coverage focuses on the structural definition of the claimed mass-tag/probe linker composition rather than on the imaging workflow limitations.

Across the claim text provided, the claimed subject matter centers on a general chemical structure with a first linker unit bearing an amine protected by Fmoc or Boc on the amine nitrogen, together with a second linker unit, which is further specified as polyethylene glycol in the dependent refinement.

Stated Advantages

Documented Applications