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公开(公告)号：US20230266214A1
公开(公告)日：2023-08-24
申请号：US17984934
申请日：2022-11-10
Applicant: Wisconsin Alumni Research Foundation
Inventor： Joshua COON , Michael WESTPHALL
CPC classification number: G01N1/42 , G01N1/36 , G01N15/1468 , H01J49/164 , H01J49/165 , H01J37/28 , H01J37/26 , H01J2237/31745
Abstract: The present invention provides methods for controllably forming a layer of amorphous ice and other amorphous solids on a substrate, and also provides cryo-electron microscopy (cryo-EM) sample preparation methods and systems that utilize in vacuo formation of amorphous ice and other solids. Formation of the amorphous solid layer can be independent of the deposition of sample molecules to be analyzed using electron microscopy, and allows for the generation of a uniformly thick layer. Optionally, mass spectrometry instruments are used to generate and purify molecules deposited on the generated amorphous solid layer. The techniques and systems described herein can deliver near ideal cryo-EM sample preparation to greatly increase resolution, sensitivity, scope, and throughput of cryo-EM protein imaging, and therefore greatly impact the field of structural biology.
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公开(公告)号：US20220365028A1
公开(公告)日：2022-11-17
申请号：US17741277
申请日：2022-05-10
Applicant: Wisconsin Alumni Research Foundation
Inventor： Jesse MEYER , Joshua COON , Alexander HEBERT , Caleb CRANNEY
IPC: G01N27/64 , H01J49/00 , G01N27/624 , G01N27/623
Abstract: The present invention provides methods and systems using gas-phase separation with mass spectrometry analysis instead of liquid chromatography, thereby enabling faster peptide, proteome, and multi-omic analysis. Also provided are improved methods and software for data independent acquisition. One embodiment referred to as Direct Infusion—Shotgun Proteome Analysis (DI-SPA) used with data-independent acquisition mass spectrometry (DIA-MS), resulted in targeted quantification of over 500 proteins within minutes of MS data collection (˜3.5 proteins/second). Enabling fast, unbiased protein and proteome quantification without liquid chromatography, DI-SPA offers a new approach to boosting throughput critical to drug and biomarker discovery studies that require analysis of thousands of proteomes. This invention is also able to perform complex multi-omic analysis of proteomes, lipidomes, and metabolomes on a single platform.
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3.发明公开公开(公告)号：US20230393041A1
公开(公告)日：2023-12-07
申请号：US18327714
申请日：2023-06-01
Applicant: Wisconsin Alumni Research Foundation
Inventor： Joshua COON , Laura MUEHLBAUER , Yunyun ZHU , Katherine OVERMYER , Annie JEN
CPC classification number: G01N1/4055 , G01N1/4044 , G01N1/38 , G01N2001/4061 , G01N2001/388
Abstract: Multi-omic analysis (analysis of proteins, lipids, and metabolites) is a powerful and increasingly utilized approach to gain insight into complex biological systems. One major hindrance with multi-omics, however, is the lengthy sample preparation process. Preparing samples for mass spectrometry (MS)-based multi-omics broadly involves extraction of metabolites and lipids with organic solvents, precipitation of proteins, and overnight digestion of proteins. The existing workflows are disparate and laborious, requiring multiple complex operation steps typically taking 1-2 days to perform. The present invention provides methods for preparing multi-omic samples that are faster and simpler than conventional methods, making it easier for a single lab or researcher to collect quality multi-omic data. A monophasic extraction solvent is used to efficiently extract biomolecules from a sample, including lipids and both polar and non-polar metabolites, and is paired with on-bead protein aggregation and rapid protein digestion.
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公开(公告)号：US20210239708A1
公开(公告)日：2021-08-05
申请号：US17231977
申请日：2021-04-15
Applicant: Wisconsin Alumni Research Foundation
Inventor： Joshua COON , Harald MARX
Abstract: In shotgun proteomics, generally only a fraction of peptides from a parent protein are actually detected. Because a large portion of the protein sequence is not detected, it is often impossible to determine whether the expressed protein is present in a modified, spliced, or truncated form. Provided herein are methods and systems for analyzing polypeptides which allow for the increase of the mean sequence coverage of a protein concomitant with bioinformatics analysis in order to distinguish putative proteoforms with improved amino acid resolution. Aspects of the invention include (1) a deep sequencing strategy to provide more protein sequence coverage than is typically achieved, and (2) a computational approach to view protein expression across its full length and identify regions of the protein that are potentially subject to such regulation. This technology has global utility in proteomics and will be of particular use for the analysis of biosimilar protein drug therapeutics.
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Patent Agency Ranking
公开(公告)号：US20220065761A1
公开(公告)日：2022-03-03
申请号：US17365698
申请日：2021-07-01
Applicant: Wisconsin Alumni Research Foundation
Inventor： Joshua COON , Michael WESTPHALL
Abstract: The present invention provides methods for controllably forming a layer of amorphous ice and other amorphous solids on a substrate, and also provides cryo-electron microscopy (cryo-EM) sample preparation methods and systems that utilize in vacuo formation of amorphous ice and other solids. Formation of the amorphous solid layer can be independent of the deposition of sample molecules to be analyzed using electron microscopy, and allows for the generation of a uniformly thick layer. Optionally, mass spectrometry instruments are used to generate and purify molecules deposited on the generated amorphous solid layer. The techniques and systems described herein can deliver near ideal cryo-EM sample preparation to greatly increase resolution, sensitivity, scope, and throughput of cryo-EM protein imaging, and therefore greatly impact the field of structural biology.
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公开(公告)号：US20180286649A1
公开(公告)日：2018-10-04
申请号：US15936288
申请日：2018-03-26
Applicant: Wisconsin Alumni Research Foundation
Inventor： Joshua COON , Nicholas RILEY , Michael WESTPHALL
Abstract: A new approach is described herein for outfitting a mass spectrometer with an infrared laser that provides an improved method of ion dissociation. One embodiment, generally referred to as Activated Ion Electron Transfer Dissociation (AI-ETD) utilizes additional energy from photons during fragmentation to generate extensive fragmentation by interacting with peptides or proteins that are not fully fragmented or separated in the high pressure linear ion trap, thus allowing for increased information during MS/MS. Additionally, a new activation scheme generally referred to as AI-ETD+ is also described that combines AI-ETD in the high pressure cell of the linear ion trap with additional infrared multi-photon dissociation (IRMPD) activation in the low pressure cell. These methods provide improved fragmentation and sequence coverage without introducing additional time to the scan duty cycle.
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7.发明公开公开(公告)号：US20240060863A1
公开(公告)日：2024-02-22
申请号：US18261267
申请日：2022-01-13
Applicant: Wisconsin Alumni Research Foundation
Inventor： Michael WESTPHALL , Joshua COON
CPC classification number: G01N1/42 , G01N1/2813
Abstract: The present invention provides an improved technique for cryogenically fixing biological samples in amorphous ice for analysis by cryo-electron microscopy (cryo-EM). Analyte particles are cooled to very low temperatures prior to depositing the particles onto a cooled substrate surface, such as a transmission electron microscope (TEM) grid. This approach “locks” in the particle structure prior to deposition. Either concurrently with or after deposition, the analyte particles are further contacted with a vapor stream of atoms or molecules at cryogenic or near cryogenic temperatures. As a result, a thin layer of an amorphous solid is formed around each particle without significant conformational changes in the particle structure, thereby forming an improved sample for EM analysis.
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公开(公告)号：US20200158607A1
公开(公告)日：2020-05-21
申请号：US16626250
申请日：2018-07-06
Applicant: Wisconsin Alumni Research Foundation
Inventor： Joshua COON , Michael WESTPHALL
Abstract: The present invention provides methods for controllably forming a layer of amorphous ice and other amorphous solids on a substrate, and also provides cryoelectron microscopy (cryo-EM) sample preparation methods and systems that utilize in vacuo formation of amorphous ice and other solids. Formation of the amorphous solid layer can be independent of the deposition of sample molecules to be analyzed using electron microscopy, and allows for the generation of a uniformly thick layer. Optionally, mass spectrometry instruments are used to generate and purify molecules deposited on the generated amorphous solid layer. The techniques and systems described herein can deliver near ideal cryo-EM sample preparation to greatly increase resolution, sensitivity, scope, and throughput of cryo-EM protein imaging, and therefore greatly impact the field of structural biology.
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公开(公告)号：US20180340941A1
公开(公告)日：2018-11-29
申请号：US15988566
申请日：2018-05-24
Applicant: Wisconsin Alumni Research Foundation
Inventor： Joshua COON , Harald MARX
Abstract: In shotgun proteomics, generally only a fraction of peptides from a parent protein are actually detected. Because a large portion of the protein sequence is not detected, it is often impossible to determine whether the expressed protein is present in a modified, spliced, or truncated form. Provided herein are methods and systems for analyzing polypeptides which allow for the increase of the mean sequence coverage of a protein concomitant with bioinformatics analysis in order to distinguish putative proteoforms with improved amino acid resolution. Aspects of the invention include (1) a deep sequencing strategy to provide more protein sequence coverage than is typically achieved, and (2) a computational approach to view protein expression across its full length and identify regions of the protein that are potentially subject to such regulation. This technology has global utility in proteomics and will be of particular use for the analysis of biosimilar protein drug therapeutics.
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