Disclosed herein, inter alia, are compositions and methods of use thereof for interrogating a cell.

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).

The invention relates to a method for quantitatively identifying relevant HLA-bound peptide antigens from primary tissue specimens on a large scale without labeling approaches. This method can not only be used for the development of peptide vaccines, but is also highly valuable for a molecularly defined immunomonitoring and the identification of new antigens for any immunotherapeutic strategy in which HLA-restricted antigenic determinants function as targets, such as a variety of subunit vaccines or adoptive T-cell transfer approaches in cancer, or infectious and autoimmune diseases.

Disclosed is a sample preparation container for purification and/or enrichment of bio-organic compounds from cellular material, viruses and/or sub-components of these. The container includes a reaction chamber and a chromatography medium. The reaction chamber is for holding the cellular material, etc. and is configured to perform reactions inside. The chromatography medium is configured to purify the bio-organic compounds. The chromatography medium is located at a wall of the reaction chamber, and the wall is closed or sealed and configured to be opened for obtaining purified bio-organic compounds. The sample preparation container further includes a receiving chamber for receiving the bio-organic compounds, that is adjacent to the chromatography medium such that the chromatography medium separates the reaction chamber from the receiving chamber. The outer face of the receiving chamber is closed and configured to be opened for obtaining purified bio-organic compounds.

The invention provides methods, reagents and systems for incorporating non-natural amino acids into proteins, preferably in vivo, using the endogenous protein synthesis machinery of an organism. The incorporated non-natural amino acids contain reactive groups for further chemical reagents, which may serve as a “handle” to enrich the proteins or fragments thereof in a number of uses, such as proteomic analysis, imaging of diseased tissues/cells, etc.

A method of mass spectrometry is disclosed for determining if a mutated variant of a target protein is present in a sample. The method comprises subjecting the sample to fragmentation so as to cause said target protein to fragment to form second generation fragment ions, and then mass analysing these fragment ions to obtain spectral data. The method determines if a mutated variant is present in the sample by determining that an ion in the spectral data has a mass to charge ratio that differs from the mass to charge ratio of an ion that would be observed if said target protein was a normal unmutated version of said target protein, and by an amount that corresponds to a mass difference that would be caused by the target protein being a mutated variant of said target protein. This method of analysing second generation fragment ions if a rapid and efficiency method of analysing a sample.

The present disclosure provides assays, such as lateral flow assays, and components thereof for detection of an analyte, e.g., a neutralizing antibody, that blocks binding of a first molecular component and a second molecular component of a molecular binding pair. In some embodiments, the disclosed assays and components thereof enable the rapid detection of a SARS-CoV-2 neutralizing antibody in a sample from an individual. Also provided in other aspects of the disclosure are devices, methods of making and using, and kits of the assays described herein.

The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the Epidermal Growth Factor Receptor (EGFR) protein that are particularly advantageous for quantifying the EGFR protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein said biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from said biological sample using the Liquid Tissue™ reagents and protocol and the EGFR protein is quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an EGFR peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

An apparatus for glycan analysis is disclosed. The apparatus includes a plurality of loading wells adapted to receive a plurality of samples; a plurality of capillaries arranged in correspondence with the loading wells, each of the capillaries including a first portion including a stacking gel and a second portion including a resolving gel; and a plurality of eluting wells arranged in correspondence with the capillaries and adapted to receive a portion of the samples having traversed the capillaries.

Disclosed herein, inter alia, are compositions and methods of use thereof for interrogating a cell.