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Publication Number
US-10101336-B2
Publication Date
2018-10-16
Expiration Date
Abstract
Microarray compositions suitable for analysis by one or several spectrographic methods are disclosed. In an embodiment, a microarray composition includes a three-dimensional solid support and a plurality of reactive microbeads positioned on the solid support in spatially distinct and addressable locations.
Core Innovation
The invention relates to automatable single-cell whole-cell mass spectrometry using microwell arrays and controlled cell-to-spot spacing relative to a mass spectrometry ionization beam spatial resolution. This approach enables reproducible whole-cell MALDI spectra and supports high-throughput analysis of very large cell numbers. It is disclosed as enabling single-cell WCMS workflows that align microwell placement with ionization and readout resolution.
The document further discloses a microwell-array device and related mass spectrometry imaging readout in which analytes are positioned with controlled localization in or around microwells. It describes microwells and a microwell-array plate suitable for live cell culture, and microbead-microwell composite microarrays with microwells supporting bead localization and analyte deposition onto defined surfaces. The deposition and elution concepts are integrated with MALDI matrix deposition approaches that include aerosol-based deposition.
In addition, the document discloses quantitative difference mass spectrometry based on reproducible whole-cell MALDI spectra for comparing treated versus control cells. It describes difference spectrum construction by spectral subtraction using a reference to enable detection of difference signals, including apoptosis detection in the presence of a drug compound. It also discloses microbead localization and recovery concepts after elution from the microwell-array format.
Claims Coverage
The provided independent claims focus on migration and elution of analytes from bead arrays using microwells, with one claim specifying aerosol exposure for migration, one specifying analytes as two chemically distinct peptides with defined differentiation criteria, and one adding a migration-then-reposition sequence. Across the independent claims, the core inventive features are bead-associated analyte migration to defined microwell surfaces and post-migration localization change by dissolving and subsequently precipitating the analyte.
Aerosol-driven analyte migration from submerged beads to defined microwell locations
Providing a bead array comprising a solid support, a bead and an analyte associated with the bead, wherein the solid support comprises a microwell and the bead is submerged in the microwell; and causing migration of the analyte from the bead onto at least one of a bottom surface of the microwell, a sidewall of the microwell, and a surface outside the microwell adjacent to the microwell, wherein the causing migration step comprises exposing the solid support to an aerosol.
Elution of two chemically distinct peptides with specified peptide differentiation criteria
Providing a bead array comprising a solid support, a bead and an analyte associated with the bead, wherein the solid support comprises a microwell and the bead is submerged in the microwell, wherein the analyte comprises two chemically distinct peptides, and wherein the two chemically distinct peptides are selected from peptides with different isotope abundance, different fluorescent labels, at least one amino acid substitution in an otherwise identical amino acid sequence, or a different number or different positions of post-translational modifications in an otherwise identical amino acid sequence; and causing migration of the analyte from the bead onto at least one of a bottom surface of the microwell, a sidewall of the microwell, and a surface outside the microwell adjacent to the microwell.
Migration followed by dissolving and precipitating to reposition analyte localization
Providing a bead array comprising a solid support, a bead and an analyte associated with the bead, wherein the solid support comprises a microwell and the bead is submerged in the microwell; causing migration of the analyte from the bead onto at least one of a bottom surface of the microwell, a sidewall of the microwell, and a surface outside the microwell that is adjacent to the microwell; and repositioning the analyte on the solid support, the repositioning step being performed after the causing migration step and comprising dissolving and subsequently precipitating the analyte on the solid support whereby localization of the analyte on the solid support changes.
The claims collectively cover submerging beads in microwells and causing analyte migration onto bottom surface, sidewall, and/or adjacent outside surface, with one claim specifying aerosol exposure for migration and another specifying analyte as two chemically distinct peptides with stated differentiation criteria. A further independent claim adds a migration-then-reposition sequence using dissolving and subsequent precipitation to change analyte localization.
Stated Advantages
High throughput analysis of very large cell numbers.
Reproducible whole-cell MALDI spectra enabling quantitative difference mass spectrometry.
Enables detection of difference signals, including apoptosis detection.
Enables localization of analytes onto defined analytical regions associated with the microwell, including the microwell bottom surface, microwell sidewall, and/or a surface outside the microwell that is adjacent to the microwell.
Enables a change in localization of the analyte on the solid support by repositioning after migration using dissolving and subsequently precipitating.
Documented Applications
Single-cell whole-cell mass spectrometry (WCMS) using microwell arrays and controlled spacing relative to ionization beam resolution.
Mass spectrometry imaging (MSI) with MALDI matrix deposition approaches and localization of analyte and matrix.
Quantitative difference mass spectrometry for comparing treated versus control cells.
Apoptosis detection using difference mass spectrometry in the presence of a drug compound.
Live cell culture using a microwell-array plate format.
Microbead-microwell composite microarray use for microbead localization and recovery after elution.
Mass spectrometry and other spectrographic readouts based on releasing analytes from bead-bound reactive sites into defined analytical regions, including MALDI and related spectrographic techniques.
Support for bead recovery, sample archiving, and kinetic measurements.
Proteomic assays.
Nucleic-acid analysis including nucleic-acid sequencing/PCR from beads.
Live/whole-cell microarrays with reproducible whole-cell mass spectrometry and difference mass spectrometry for comparing cell populations.
Magnetic-bead handling.
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