Photocleavable Mass Tags for Multiplex MSI Without Spectral Overlap
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Solution Overview
Problem
Current methods for multiplex immunohistochemistry and in situ hybridization, such as fluorescence microscopy and mass spectrometric imaging, face limitations in simultaneously detecting multiple biomarkers due to spectral overlap and sensitivity issues, particularly in identifying intact proteins and nucleic acids, which restricts the ability to unravel complex biological systems and human diseases.
Innovation Solution
The development of photocleavable mass-tag reagents attached to probes like antibodies and nucleic acids, enabling high-plex mass spectrometric imaging (MSI) that combines targeted and untargeted analysis, allowing for simultaneous detection of multiple biomarkers on a single specimen through novel photocleavable peptide mass-tags (PC-MTs) and MALDI-MSI procedures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If fluorescence microscopy is used for multiplex immunohistochemistry, then simultaneous detection of multiple biomarkers is achieved, but spectral overlap limits the number of detectable biomarkers to only 3-5
Solution Approach 1:
The patent replaces optical detection (fluorescence microscopy) with mass spectrometric detection. Instead of using fluorophores that emit light at different wavelengths, the invention uses mass-tags with distinct mass-to-charge ratios that are detected by mass spectrometry, eliminating spectral overlap and enabling simultaneous detection of many more biomarkers (multiplexing beyond 3-5 targets).
Solution Approach 2:
The invention changes the detection parameter from optical wavelength (fluorescence emission spectrum) to mass-to-charge ratio. By using mass-tags with carefully selected distinct masses rather than fluorophores with distinct emission wavelengths, the system achieves higher multiplexing capacity since mass spectrometry can resolve many more discrete mass values than there are non-overlapping fluorescence spectra.
2Measurement precision
If mass spectrometric imaging is used for detecting intact proteins and nucleic acids, then targeted detection capability is improved, but sensitivity is insufficient for robust detection
Solution Approach 1:
The patent introduces mass-tags as intermediary molecules that are attached to probes (antibodies or nucleic acid probes). These mass-tags serve as detectable surrogates that mediate between the probe-target binding event and the mass spectrometric detection. The mass-tags are designed to be highly ionizable and produce strong signals, thereby amplifying the detection sensitivity while maintaining the targeted detection capability for intact biomolecules.
Solution Approach 2:
The invention modifies the detection parameters by using specific mass-tag compositions and ionization conditions that optimize signal intensity. The mass-tags are designed with chemical properties that enhance ionization efficiency in MALDI-MS, and the use of matrix-assisted laser desorption ionization with optimized matrix compounds improves the sensitivity and reliability of detecting intact proteins and nucleic acids.
3Quantity of substance
If photocleavable mass-tags are used for multiplexed MSI, then simultaneous detection of multiple biomarkers is enabled, but additional photocleavage step increases procedural complexity
Solution Approach 1:
The patent applies photocleavage to release the mass-tags from the probes before mass spectrometric imaging. This preliminary action separates the detection signal (mass-tag) from the probe molecule, allowing the mass spectrometer to detect only the mass-tags with high sensitivity and resolution. The photocleavage step, while adding a procedural element, enables the mass-tags to be freely detected without interference from the larger probe molecules, thereby facilitating high-plex multiplexing.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach achieves highly multiplexed and multi-omic tissue imaging, overcoming previous limitations by enabling robust sensitivity and simultaneous detection of a wide range of biomarkers, including proteins and nucleic acids, on various tissues, while correlating with conventional fluorescence imaging.
Implementation Method 1
Photocleavable mass-tag reagents attached to probes like antibodies and nucleic acids
Implementation Method 2
detecting, using mass spectrometric imaging, said mass-tags, or fragments thereof, as molecular ions
Implementation Method 3
MALDI-MSI procedures
Data Source
AI summary
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. 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, and also encompasses multi-omic MSI procedures, where MSI of photocleavable mass-tag probes is combined with other modes of MSI.