WO2023025318A1 - Methods and formulations for expansion proteomic analysis of biological samples - Google Patents

Methods and formulations for expansion proteomic analysis of biological samples Download PDF

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WO2023025318A1
WO2023025318A1 PCT/CN2022/115456 CN2022115456W WO2023025318A1 WO 2023025318 A1 WO2023025318 A1 WO 2023025318A1 CN 2022115456 W CN2022115456 W CN 2022115456W WO 2023025318 A1 WO2023025318 A1 WO 2023025318A1
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sma
hydrogel
sample
dmaa
pae
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PCT/CN2022/115456
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French (fr)
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Piatkevich KIRYL
Tiannan GUO
Cuiji SUN
Lu Li
Yaoting SUN
Yi Zhu
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Westlake University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers

Definitions

  • the present disclosure relates to proteomics, especially a method of proteomics analysis of tissue samples in combination with hydrogel expansion technology, specific formulations used in such methods and uses thereof, as well as kits and systems used for such methods.
  • Protein subcellular positioning is closely related to the functions of proteins, but traditional high-throughput research methods such as transcriptome sequencing cannot be used for such studies. With the development of microscopy, mass spectrometry and machine learning methods, the study of protein subcellular positioning and its dynamic changes through spatial proteomics is becoming an important tool for understanding cell biology.
  • One of the spatial proteomics research methods is to analyze protein positioning based on imaging without the need to perform cell lysis or isolation of cellular compartments or organelles before proteomics analysis.
  • Protein imaging is one of the interested areas in the development of imaging mass spectrometry (IMS) technology.
  • IMS imaging mass spectrometry
  • MALDI Microx Assisted Laser Desorption Ionization
  • MS technology developed based on this is capable of multiplex analyzing thousands of analytes on the surface of the samples, to obtain a two-dimensional molecular map that reveals the positions and relative abundance of the molecules, especially allowing the visualization of protein and various protein forms.
  • MALDI has limited use due to its relatively low accuracy.
  • the main solution adopted by the spatial proteomics research of biological tissue samples is to use laser capture microdissection (LCM) methods to obtain tiny tissue samples.
  • LCM can extract a single cell sample from a heterogeneous environment via a microscope combined with a laser aiming system, use lasers to perforate the tissue and remove target area, and separate it from adjacent tissue areas.
  • the polypeptide fragment is obtained through laser sampling and traditional in-solution trypsin digestion, and then rendered for proteomics analysis.
  • LCM sampling can only obtain samples of about 3-5 mm, and the price of LCM is very expensive and difficult for large scale application; traditional in-solution trypsin digestion for treating the obtained samples would lose a lot of low-abundance proteins during the treatment.
  • the present disclosure solves the problem of effectively obtaining tiny tissue samples, and achieves the analysis of micro-spatial proteomics, the technology that we refer to herein as ProteomEx.
  • the present disclosure provides a method of reversibly anchoring a biological sample to a hydrogel and uniformly expanding the formed sample-hydrogel composite. After the biological sample-hydrogel composite are stained and expanded, a visualized tissue sample linearly expanded by about 6 folds can be obtained. Subsequently, tiny tissue samples with original diameter of about 500 microns can be obtained by using a puncher and used for proteomics identification.
  • the present disclosure provides a method for proteomic identification of a biological sample, comprising:
  • the biological sample is selected from a specimen, a tissue slice, a biopsy sample, an organ or part thereof, and an entire organism.
  • the sample can be live, fixed or preserved, such as a fixed tissue slice, a fresh frozen tissue or a tissue slice embedded in paraffin.
  • the sample to be expanded is a tissue slice with a thickness of 50 ⁇ m or less, i.e. a thin tissue sample. In some other embodiments, the sample to be expanded is a tissue sample with a thickness of more than 400 ⁇ m. In some other embodiments, the sample to be expanded is an entire organ.
  • the method comprises immunostaining (such as antibody staining) of the sample before treated with the anchoring agent.
  • immunostaining is performed simultaneously with anchoring.
  • the anchoring agent comprises a biomolecule reactive chemical group and a hydrogel reactive chemical group.
  • the anchoring agent can be selected from acrylate-N-succinimidyl ester (NSA) , N- (allyloxycarbonyloxy) -succinimide (NAS) and combinations thereof.
  • the anchoring includes contacting the biological sample with an anchor agent, then infusing the biological sample with a hydrogel precursor solution or embedding the biological sample in the hydrogel precursor solution.
  • the anchoring includes infusing the biological sample with a hydrogel precursor solution added with the anchoring agent or embedding the biological sample in the hydrogel precursor solution added with the anchoring agent, thus anchoring and polymerization are performed simultaneously.
  • the hydrogel precursor solution comprises a precursor (s) of the hydrogel polymer.
  • the precursor (s) is/are selected from sodium acrylate (SA) , sodium methacrylate (SMA) , itaconic acid (IA) , trans-aconitic acid (TAA) , ethyl-2- (hydroxymethyl) -acrylate (EHA) , N, N-methylenebisacrylamide (MBAA, also known as BIS) , N, N-dimethylacrylamide (DMAA) , pentaerythritol tetraacrylate (PT) , propoxylated trimethylol propane triacrylate (TPT) , pentaerythritol triacrylate (PA) , dipentaerythritol pentaacrylate or dipentaerythritol hexaacrylate (DPHA) , trimethylolpropane triacrylate (TTA) , bis (trimethylol) propane) -tetraacryl
  • the precursors comprise SMA, DMAA and PAE.
  • the molar ratio of DMAA: SMA included in the precursors is in the range of 30: 1 to 3: 1, e.g., in the range of 20: 1 to 3: 1, in the range of 15: 1 to 3: 1, in the range of 10: 1 to 3: 1, in the range of 9: 1 to 3: 1, in the range of 8: 1 to 3: 1, in the range of 7: 1 to 3: 1, in the range of 6: 1 to 3: 1, in the range of 5: 1 to 3: 1, in the range of 4: 1 to 3: 1, or any sub-range thereof, more specifically, the molar ratio of DMAA: SMA may be about 4: 1 or 3: 1; and wherein the molar ratio of SMA: PAE is in the range of 10: 0.005 to 10: 3, such as 10: 0.008 to 10: 3, 10: 0.01 to 10: 3, 10: 0.02 to 10: 3, 10: 0.03 to 10: 3, 10
  • the molar ratio of DMAA: SMA: PAE contained in the precursors is in the range of (40 ⁇ 20) : (10 ⁇ 5) : (0.005-2) , such as about (40 ⁇ 15) : (10 ⁇ 3.5) : (0.005-2) , about (40 ⁇ 10) : (10 ⁇ 2.5) : (0.005-2) , about (40 ⁇ 5) : (10 ⁇ 2) : (0.005-2) , about (40 ⁇ 5) : (10 ⁇ 1.2) : (0.005-2) , about (40 ⁇ 5) : (10 ⁇ 1) : (0.005-2) , preferably about 40: 10: (0.005-2) , about 40: 10: (0.006-2) , about 40: 10: (0.008-2) , about 40: 10: (0.008-1.8) , about 40: 10: (0.05-1.8) , about 40: 10: (0.05-1.6) , about 40: 10: (0.05-1.4) , about 40: 10: (0.08-1.4) , including any sub-ranges thereof.
  • the precursors comprise SMA, DMAA and TPT.
  • the molar ratio of DMAA: SMA included in the precursors is in the range of 30: 1 to 3: 1, e.g., in the range of 20: 1 to 3: 1, in the range of 15: 1 to 3: 1, in the range of 10: 1 to 3: 1, in the range of 9: 1 to 3: 1, in the range of 8: 1 to 3: 1, in the range of 7: 1 to 3: 1, in the range of 6: 1 to 3: 1, in the range of 5: 1 to 3: 1, in the range of 4: 1 to 3: 1, or any sub-range thereof, more specifically, the molar ratio of DMAA: SMA may be about 4: 1 or 3: 1; and wherein the molar ratio of SMA: TPT is in the range of 10: 0.005 to 10: 3, such as 10: 0.008 to 10: 3, 10: 0.01 to 10: 3, 10: 0.02 to 10: 3, 10: 0.03 to 10: 3, 10
  • the molar ratio of DMAA: SMA: TPT contained in the precursors is in the range of (40 ⁇ 20) : (10 ⁇ 5) : (0.005-2) , such as about (40 ⁇ 15) : (10 ⁇ 3.5) : (0.005-2) , about (40 ⁇ 10) : (10 ⁇ 2.5) : (0.005-2) , about (40 ⁇ 5) : (10 ⁇ 2) : (0.005-2) , about (40 ⁇ 5) : (10 ⁇ 1.2) : (0.005-2) , about (40 ⁇ 5) : (10 ⁇ 1) : (0.005-2) , preferably about 40: 10: (0.005-2) , about 40: 10: (0.006-2) , about 40: 10: (0.008-2) , about 40: 10: (0.008-1.8) , about 40: 10: (0.05-1.8) , about 40: 10: (0.05-1.6) , about 40: 10: (0.05-1.4) , about 40: 10: (0.08-1.4) , including any sub-ranges thereof.
  • the hydrogel precursor solution also comprises a polymerization activation agent and/or polymerization promoting agent (preferably added before use) , such as VA-044, V50, ammonium persulfate, potassium persulfate and TEMED.
  • a polymerization activation agent and/or polymerization promoting agent preferably added before use
  • the sample-hydrogel composite is homogenized by denaturation, preferably a mild denaturation.
  • the protein degeneration is performed by treating with a detergent (such as SDS or sodium dodecyl benzene sulfonate) .
  • the method comprises treating the biological sample with a disulfide bond reductant, and the disulfide bond reductant is e.g. selected from TCEP, DTT, and BME.
  • the disulfide bond reduction may be performed before anchoring.
  • the method further comprises de-anchoring before protein staning.
  • de-anchoring may be performed by increasing pH value to alkaline (for example, by contacting Tris buffer) to break the amide bond.
  • the staining is Coomassie blue staining.
  • the expansion of the sample-hydrogel composite is performed by incubating the composite with water or an aqueous buffer.
  • the linear extension factor of the expanded sample-hydrogel composite may be 2-7, for example above 5.
  • the sampling is local sampling, such as micro-dissection or local sampling via a punch.
  • the diameter of the samples obtained from local sampling can be less than 3 mm.
  • the diameter of the samples obtained by micro-dissection may reach micrometer grade.
  • protein identification is performed by mass spectrometry analysis, including enzymatic digestion, peptide extraction (optionally demineralization) and mass spectrometry detection on the sample-hydrogel composite.
  • Mass spectrometry detection can be performed via HPLC-MS or MALDI-TOF.
  • the disclosure provides a hydrogel formulation comprising a combination of DMAA, SMA and PAE, or a combination of DMAA, SMA and TPT.
  • the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1
  • the molar ratio of SMA: PAE is within the range of 10: 0.01 to 10: 3
  • the molar ratio of DMAA: SMA: PAE is in the range of e.g.
  • the molar ratio of DMAA: SMA: PAE can be about 30: 10: 1, 40: 10: 0.008, 40: 10: 0.4, 40: 40
  • the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1
  • the molar ratio of SMA: TPT is within the range of 10: 0.01 to 10: 3
  • the molar ratio of DMAA: SMA: TPT is in the range of e.g.
  • the molar ratio of DMAA: SMA: TPT can be about 30: 10: 1, 40: 10: 0.008, 40: 10: 0.4, 40,
  • the hydrogel formulation is prepared as a stock solution of DMAA, SMA, and PAE diluted in water, or DMAA, SMA and TPT diluted in water.
  • the hydrogel formulation is prepared as a mixture of DMAA, SMA, and PAE without dilution, or DMAA, SMA and TPT without dilution.
  • the disclosure provides use of the hydrogel formulation as disclosed herein for expansion of a biological sample, wherein the hydrogel formulation comprises hydrogel precursors comprising DMAA, SMA and PAE, or DMAA, SMA and TPT.
  • the disclosure provides a kit, comprising DMAA, SMA and PAE in one or more containers, or DMAA, SMA and TPT in one or more containers.
  • DMAA, SMA, and PAE are each in separated containers, respectively.
  • the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1
  • the molar ratio of SMA: PAE is within the range of 10: 0.005 to 10: 3.
  • at least two of DMAA, SMA, and PAE are mixed in one container. The ratios of DMAA, SMA, and PAE may be as described as above.
  • DMAA, SMA, and TPT are each in separated containers, respectively.
  • the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1
  • the molar ratio of SMA: TPT is within the range of 10: 0.005 to 10: 3.
  • at least two of DMAA, SMA, and TPT are mixed in one container. The ratios of DMAA, SMA, and TPT may be as described as above.
  • the kit is suitable for storage at 0°C-4°C or at room temperature.
  • the kit further contains one or more containers comprising NAS and/or NSA as anchor agents.
  • the kit further contains one or more of the following:
  • a container comprising a disulfide bond reductant such as TCEP
  • a container comprising a fixative such as paraformaldehyde
  • a container comprising a protein denaturation buffer such as a protein denaturation buffer comprising SDS
  • a container comprising protein staining solution such as Coomassie blue staining buffer
  • a container comprising enzymes such as trypsin for digestion of proteins in the sample
  • a container comprising one or more buffers such as TCEP
  • the kit further comprises one or more of the following: a gelation room configured to accommodate the sample before gelation, such as a dish with cavity or grooves; a staining room configured to stain the proteins in the sample; a hydrogel operation tool, such as a tweezer, soft brush; a puncher; and a cooling device, such as ice bags.
  • a gelation room configured to accommodate the sample before gelation, such as a dish with cavity or grooves
  • a staining room configured to stain the proteins in the sample
  • a hydrogel operation tool such as a tweezer, soft brush
  • a puncher such as ice bags.
  • This disclosure also provides the kit as described above for physical expansion of biological samples.
  • the disclosure provides a system of spatial proteomics analysis of a biological sample, including:
  • a system for spatial proteomics analysis of a biological sample comprising:
  • an anchoring module for anchoring the proteins in the biological sample
  • a gelation module for sample anchoring and hydrogel polymerization to form a sample-hydrogel composite
  • a protein degeneration and de-anchoring module for denaturation and de-anchoring of the proteins in the sample-hydrogel composite respectively;
  • a staining module for protein staining and optionally local sampling of the sample-hydrogel composite
  • mass spectrometry analysis module for pre-processing of the samples obtained by local sampling before mass spectrometry and which is connected with a mass spectrometry detection device.
  • the system further comprises a protein pre-processing module for disulfide bond reduction of the biological sample, and/or immune-staining.
  • one or more of the modules can be integrated together.
  • the anchoring module and gelation module can be integrated into one module, thereby anchoring and gelation are performed at the same time.
  • FIG. 1 illustrates ProteomEx workflow.
  • A Chemically fixed tissue samples, which can be immunostained beforehand, are treated with the chemical anchor, embedded into the hydrogel, and mechanically homogenized by mild denaturation.
  • the Coomassie-stained (CBB) hydrogel embedded samples are expanded and imaged. After imaging, expanded samples are microdissected and excised pieces of the tissue-hydrogel composite are processed by in-gel digestion to recover peptides for LC-MS/MS analysis (acronyms LEF, linear expansion factor, VOL, volumetric expansion factor) .
  • B Timeline of ProteomEx indicating total duration and hands-on time of each step (total duration/hands-on time) .
  • C Brightfield images of mouse brain tissue section before expansion and
  • D after Coomassie staining and expansion
  • Figure 2 illustrates a schematic diagram of manual local sampling of a brain slice sample-hydrogel composite after expansion using a 3 mm punch, the left panel is the sample before punching, and the right panel is the sample after punching.
  • Figure 3 illustrates the size of the brain slice sample-hydrogel composite before and after swelling, the left panel is the size of the sample before swelling, the right panel is the size of the sample after swelling. It can be seen that the brain slice swelled about 6.1 times.
  • Figure 4 illustrates a graph of the number of protein species obtained by proteomics identification after the expanded gel was punched with a 3 mm puncher for sampling.
  • Figure 5 illustrates a histogram of the number of identified protein species (A) and the proportion of missing cleavage (B) for brain slice samples subjected to swelling gel digestion, in-solution digestion, and pressure cycling digestion (from left to right) .
  • Figure 6 illustrates ProteomEx benchmarking against standard methods in the field.
  • B Missed cleavages of the identified peptides prepared using in-solution, PCT, proExM-MS, and ProteomEx methods.
  • E Venn diagram of identified proteins for the bulk samples shown in D.
  • F The subcellular locations of the identified proteins for the samples shown E.
  • Figure 7 illustrates reproducibility and stability comparison for the four methods.
  • A Heatmap of Pearson correlations of each paired samples from the four strategies analyzed using the MSFragger software. The color bar indicates the values of Pearson correlations.
  • B Coefficient of variation of quantified protein abundance from the four methods.
  • Figure 8 illustrates peptide modifications analysis.
  • A The upset plot showing the number of overlapped modifications identified in the four methods by open search mode.
  • B Distribution of different modifications ranked by peptide modification ratio from ProteomEx. The four color indicate corresponding methods.
  • Figure 9 illustrates validation of ProteomEx in different tissue types.
  • A Representative bright-field images of different tissue slices pre-expansion (left column) and post-expansion (middle column; Coomassie-stained) and overlay (right column) of pre-expansion image (green pseudo-color) and registered post-expansion image (magenta pseudo-color) .
  • (E) Number of peptide and protein identifications for 0.37 nL brain tissue analyzed in PulseDIA mode (n 3 punches from one slice) .
  • tissue samples with a diameter of about 100-1000 (e.g. 128-1000) microns can be manually obtained to determine accurate spatial positioning of the proteins in the tissue sample; by anchoring the proteins to the hydrogel, the problem of losing a large amount of proteins in the traditional in-solution trypsin digestion method can be avoided; the protein anchored in the hydrogel can be more easily digested by trypsin after denaturation, lipid removal and swelling, obtaining more effective peptide fragments; tiny tissue samples treated with this protocol can identify more proteins than traditional in-solution trypsin digestion and pressure cycling technology of the second generation.
  • the chemical reagents and processing conditions used in the methods of the present disclosure are more economic and easily available, thus more suitable for large-scale applications and accessible for an average biochemical and analytical laboratories and facilities.
  • the term “expand” , “expansion” , “swell” , “magnification” , “swelling” and their grammatical variations generally refer to expansion or enlargement, e.g., uniform expansion in 3 dimensions, that occurs upon contact with a liquid such as water or other solvents.
  • the expandable material is preferably transparent so that light can pass through the sample when expanded.
  • the swellable material may be a hydrogel.
  • the expandable material is formed from the polymerization of its precursors, monomers or oligomers.
  • the monomer may contain a water-soluble group containing a polymerizable ethylenically unsaturated group.
  • the monomer or oligomer may comprise one or more of the following: substituted or unsubstituted methacrylates, acrylates, acrylamides, methacrylamides, vinyl alcohols, vinylamines, allylamines, allyl alcohols, including its divinyl crosslinkers (e.g., N, N-alkylene bisacrylamides) .
  • the expandable material is polyacrylates and copolymers or cross-linked copolymers thereof.
  • the swellable material can be formed in situ by chemically crosslinking water-soluble oligomers or polymers. In the present invention, it is contemplated that a precursor solution of a swellable material is added to the sample, allowing the precursor to polymerize and swell in situ.
  • the term "Expansion proteomics” refers to a process of reversible anchoring of biological samples (usually tissue samples) to hydrogels that can be uniformly swelled, and the formed sample-hydrogel composite is subjected to protein denaturation, de-anchoring, staining, swelling, sampling and enzymatic digestion to obtain polypeptides for proteomic analysis (especially spatial proteomics analysis) .
  • the present disclosure provides a method for the spatial proteomics analysis of proteins from spatially distinct regions of a biological sample (e.g. a tissue section) .
  • the method comprises polymerizing a sample with hydrogel precursors to form a sample-hydrogel composite, staining proteins in the sample, swelling the sample-hydrogel composite, and acquiring micro-samples from the expanded composite for protein identification.
  • the protein identification includes identification of peptides sampled and extracted from the composite by mass spectrometry techniques (eg, LC-MS/MS) .
  • the present disclosure provides a method of expanding a tissue sample to obtain tiny tissue samples (e.g. at ⁇ m grade) , wherein a sample-hydrogel composite is formed in a manner that preserves the relative positions of biomolecules (e.g. proteins) in their original functional status. Since the relative positions of the proteins are preserved, the localization information of the proteins at different positions in the sample can be acquired by sampling different regions of the gel and identifying the proteins.
  • biomolecules e.g. proteins
  • the method as disclosed herein comprises one or more of the following steps:
  • pre-processing the biological sample for example immunostaining or treating the sample with a disulfide bond reductant;
  • the present disclosure also contemplates de-swelling the sample-hydrogel complex, e.g. returning to its original size, by soaking in a high salt buffer. Therefore, the method of the present invention is able to reversibly modulate the expansion factor of the sample-hydrogel composite.
  • the present disclosure also exemplifies some chemical anchoring agents or combinations thereof that can be used for protein anchoring, which can more rapidly penetrate larger biological samples to anchor biomolecules.
  • the methods herein are applicable not only to cells and thin samples, but also to large or thick samples.
  • the hydrogel obtained in the methods herein has high mechanical stability after expansion, allowing its application to expand large tissue blocks, tissue slices, whole organs and even whole organisms without deforming and mechanically disintegrating under its own weight, which show obvious advantages compared to other hydrogels.
  • the hydrogel precursor solution or hydrogel formulation from which the hydrogel is formed comprises DMAA, SMA, and PAE.
  • the methods of the present disclosure can be used for proteomic (especially spatial proteomic) studies of different organs and tissues, such as brain mapping, connectome studies as well as new diagnostics, personalized medicine, histopathology and other medical applications.
  • Biological samples for hydrogel anchoring and expansion in the present disclosure can be fresh, frozen, or fixed (i.e. preserved) .
  • the source of biological samples can be a solid tissue, for example from a fresh, frozen and/or preserved organ, or a tissue sample or biopsy or aspiration sample, including preserved tumor samples such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.
  • a tissue sample may be one that is routinely prepared and preserved in routine clinical practice.
  • Fixed samples are histologically preserved samples using a fixative (e.g., formaldehyde, paraformaldehyde) . Fixation can be accomplished before, during, or after the disulfide bond reduction treatment, protein anchoring, and gelation. In certain embodiments, the sample of interest is fixed prior to disulfide bond reduction treatment.
  • Samples can also be embedded in solid and generally rigid media such as paraffin, wax, collodion or resin, so that thin sections can be cut for processing.
  • Fixation can be carried out by conventional methods. Those skilled in the art will understand that the choice of fixative will depend on the purpose for which the sample is to be stained histologically or otherwise analyzed. Those skilled in the art will also understand that the time of fixation depends on the size of the tissue sample and the fixative used. For example, neutral buffered formalin, Bouin or paraformaldehyde can be used to fix the sample.
  • the tissue sample is a thin tissue section (as thin as 5 ⁇ m) , such as a brain section, liver section, or tumor section.
  • the sample used for expansion is a tissue sample with a thickness greater than 400 ⁇ m or even a whole organ and parts thereof, the organ including but not limited to brain, spinal cord, heart, lung, liver, kidney, stomach, colon, bone, muscle, skin, lymph nodes, genitals, breast, pancreas, prostate, bladder, thyroid and eyes, etc.
  • a disulfide bond is a chemical bond that connects the sulfhydryl groups of two cysteine residues in different peptide chains of a protein or in the same peptide chain.
  • Disulfide bonds are relatively stable covalent bonds, which play a role in stabilizing the spatial structure of peptide chains in protein molecules. The larger the number of disulfide bonds, the greater the stability of the protein molecule against the influence of external factors. Therefore, in order to promote the anchoring of proteins and hydrogels in the subsequent process, a disulfide bond reduction treatment may be performed on the sample to destroy the disulfide bonds of protein molecules therein.
  • Disulfide bonds in the sample can be broken by treatment with reducing agents.
  • reducing agents There are a variety of reducing agents known in the art to achieve disulfide bond cleavage, commonly used reducing agents are thiols such as ⁇ -thioethanol ( ⁇ -ME) or dithiothreitol (DTT) . Excess thiol reagent is usually used to ensure complete cleavage of the disulfide bonds.
  • Other reducing agents include TCEP. Unlike ⁇ -ME and DTT, TCEP is odorless, selective, can work in alkaline and acidic environments (unlike DTT) , is more hydrophilic, and is resistant to oxidation.
  • the biological sample is incubated with a reducing agent for a sufficient period of time for the disulfide bond reduction treatment, wherein the reducing agent is selected from the group consisting of TCEP, ⁇ -ME and DTT.
  • the reducing agent can be formulated as a solution, for example, a solution of TCEP can be prepared by dissolving the hydrochloride salt form of TCEP in boric acid buffer.
  • the sample is contacted with a chemical anchoring agent or perfused with an anchoring agent solution, and is incubated for a sufficient time for the chemical reaction to occur.
  • the terms “anchoring agent” and “crosslinking agent” are used interchangeably herein.
  • the chemical anchoring agent is preferably a bifunctional anchor comprising a biomolecule reactive chemical group and a hydrogel reactive chemical group.
  • chemical anchoring agents containing one or more functional groups attach reactive groups to functional groups (e.g., primary amines or sulfhydryls) of biomolecules within the biological sample, which may be proteins, nucleic acids, lipids, proteoglycans, lipopolysaccharides, etc. Since the present disclosure is focused on proteomics study, the anchoring agents employed are those that are preferred towards anchoring proteins.
  • the anchoring agent functionalizes biomolecules such as proteins in the sample to react with the growing chains of the hydrogel polymer upon polymerization of the hydrogel, thereby covalently anchoring the functionalized biomolecules to the hydrogel network.
  • Chemical anchoring agents are usually provided in the form of anchoring agent solutions.
  • the anchoring agent solution is prepared by dissolving or diluting the chemical anchoring agent in an aqueous buffer such as PBS, MES buffer.
  • the anchoring agent solution may also contain a fixing agent to fix the sample at the same time as anchoring.
  • a fixing agent to fix the sample at the same time as anchoring.
  • One skilled in the art can easily determine the buffers for preparing the anchoring agent solution based on the selected anchoring agent.
  • the chemical anchoring agent comprises two distinct functional groups, one for functionalization of biomolecules, particularly protein molecules, and the other for reaction with growing polymer chains. Functionalization of biomolecules can be performed during chemical fixation, i. e., on live or freshly frozen samples, or after chemical fixation, on fixed and preserved samples. As understood by those skilled in the art, the two distinct functional groups of a bifunctional chemical anchor can be separated by a chemical spacer of various lengths and structures, including branched spacers.
  • a biomolecule reactive chemical group includes, but not limited to, N-hydroxysuccinimide (NHS) esters, epoxy groups, aldehyde groups, carboxamide groups, which can react with amino or carboxylic acid groups on proteins, peptides, nucleic acids and/or lipids.
  • a hydrogel reactive group includes, but not limited to, vinyl, allyl, acrylate, methacrylate, acrylonitrile, acrylamide groups.
  • the chemical anchoring agent used to directly crosslink the protein to any hydrogel polymer chain is selected from the group consisting of acrylate-N-succinimidyl ester (NSA) , N- (allyloxycarbonyloxy) -succinimide (NAS) , and a combination thereof.
  • NSA acrylate-N-succinimidyl ester
  • NAS N- (allyloxycarbonyloxy) -succinimide
  • Treatment with NSA or NAS modifies the amine on the protein with acrylamide or allyloxycarbonyloxyamide functional group, respectively, allowing the functionalized protein to anchor to the hydrogel during polymerization.
  • the chemical anchoring agent comprises NSA.
  • the anchoring agent comprises NAS.
  • the anchoring agent comprises both NAS and NSA, e.g., NAS and NSA at a 1: 1 molar ratio.
  • the anchoring agent can be diluted in the anchoring buffer at various ratios, e.g., about 1: 10, 1: 20, 1: 30, 1: 40, 1: 50, 1: 100, 1: 150, 1: 200, depending on the choice of buffer and anchoring agent, desired reaction rate and anchoring effect, among others.
  • a biological sample is permeated (e.g., infused, injected, impregnated, added, or mixed in other ways) or embedded in a hydrogel precursor solution, and then polymerization takes place to form a swellable sample-hydrogel composite.
  • the composite comprises a swellable polymer network in which protein molecules within the sample (either already functionalized or being functionalized by an anchor) are covalently bound to the polymer chains of the hydrogel.
  • Hydrogel precursor solutions typically comprise one or more hydrogel monomers, oligomers or precursors dissolved in an aqueous solution such as water or an aqueous solution added with an acid to adjust pH.
  • aqueous solution such as water or an aqueous solution added with an acid to adjust pH.
  • the terms "monomer” or “precursor” in relation to hydrogel are used interchangeably herein to include monomers, co-monomers, and crosslinkers that build the small units of the hydrogel polymer chain.
  • the hydrogel precursor solution can also be added with polymerization activators or accelerators such as VA-044, V50, APA, potassium persulfate and TEMED prior to use. Water or an aqueous solution is typically used as the dispersion medium in the forming hydrogel precursor solution, but other solvents can also be used.
  • the hydrogel precursor will polymerize/cross-link with the sample to form a hydrogel-sample composite.
  • the precursors of hydrogel include, but are not limited to, sodium acrylate (SA) , sodium methacrylate (SMA) , itaconic acid (IA) , trans-aconitic acid (TAA) , ethyl-2- (hydroxymethyl) -acrylate (EHA) , N, N-methylenebisacrylamide (MBAA, also known as BIS) , N, N-dimethylacrylamide (DMAA) , pentaerythritol tetraacrylate (PT) , propoxylated trimethylol Propane triacrylate (TPT) , pentaerythritol triacrylate (PA) , dipentaerythritol pentaacrylate or dipentaerythritol hexaacrylate (DPHA) , trimethylolpropane triacrylate (TTA)
  • SA
  • the sample of interest is to be embedded in a sodium polyacrylate based hydrogel
  • the sample may be perfused with a solution comprising the monomers sodium acrylate and acrylamide as well as the anchoring agent N, N-methylenebisacrylamide.
  • a polymerization accelerator or initiator is added to the solution prior to infusion, so that upon permeation of the sample the solution is also activated to form the hydrogel polymer.
  • such monomer-containing solutions are aqueous.
  • the precursor of the hydrogel can be selected from N, N-dimethylacrylamide (DMAA) , sodium methacrylate (SMA) and pentaerythritol allyl ether (PAE) , preferably comprising all three.
  • the precursor of the hydrogel can be selected from N, N-dimethylacrylamide (DMAA) , sodium methacrylate (SMA) and propoxylated trimethylol Propane triacrylate (TPT) , preferably comprising all three.
  • DMAA, SMA and PAE are selected as precursors, wherein the molar ratio of DMAA to SMA is in the range of 30: 1 to 3: 1, such as in the range of 20: 1 to 3: 1, in the range of 15: 1 to 3: 1, in the range of 10: 1 to 3: 1, in the range of 9: 1 to 3: 1, in the range of 8: 1 to 3: 1, in the range of 7: 1 to 3: 1, in the range of 6: 1 to 3: 1, in the range of 5: 1 to 3: 1, in the range of 4: 1 to 3: 1, more specifically, the molar ratio of DMAA: SMA may be about 4: 1 or 3: 1; and wherein the molar ratio of SMA: PAE is in the range of 10: 0.005 to 10: 3, e.g., in the range of 10: 0.05 to 10: 3, 10: 0.08 to 10: 3, 10: 0.1 to 10: 3, 10: 0.2 to 10: 3, 10: 0.3 to 10: 3, 10: 0.4
  • the precursors comprise DMAA, SMA and PAE, and the molar ratio of DMAA: SMA: PAE is in the range of (40 ⁇ 20) : (10 ⁇ 5) : (0.005-2) , such as about (40 ⁇ 15) : (10 ⁇ 3.5) : (0.4 ⁇ 0.2) , about (40 ⁇ 10) : (10 ⁇ 2.5) : (0.4 ⁇ 0.2) , about (40 ⁇ 5) : (10 ⁇ 2) : (0.4 ⁇ 0.2) , about (40 ⁇ 5) : (10 ⁇ 1.2) : (0.4 ⁇ 0.2) , about (40 ⁇ 5) : (10 ⁇ 1) : (0.4 ⁇ 0.1) .
  • the precursor comprises DMAA, SMA, and PAE, wherein, on a mole basis, if DMAA is 20-60 parts, SMA is 5-15 parts, and PAE is 0.005-1.5 parts.
  • the molar ratio of DMAA: SMA: PAE can be about 30: 10: 1, 40: 10: 0.008, 40: 10: 0.4, 40: 10: 1, 40: 10: 1.2, or 40: 10: 1.5.
  • the precursor solution can be formulated by adding water or an aqueous solution (e.g., an aqueous solution with the addition of an acid to adjust the pH) to the hydrogel precursor or by dissolving the hydrogel precursor in water or an aqueous solution.
  • concentration of the hydrogel precursor contained in the precursor solution may be in the range of 30-60%on a weight basis (w/w) , such as 30%, 40%, 50%or 60%.
  • Stock solutions of hydrogel precursor solutions can be prepared and diluted prior to use. Stock solutions may have high concentrations (w/w) of precursors, e.g., about 50%or higher, about 75%or higher, about 80%or higher, about 90%or higher.
  • the stock solution can be diluted to a concentration of about 30-50%when applied to the sample.
  • the obtained hydrogel has superior mechanical strength and expansion factor properties.
  • the precursors can be mixed in various ratios to form hydrogels that swell adjustably and reversibly in aqueous buffers at an expansion factor of up to 7-fold or more in the linear dimension.
  • DMAA: SMA: PAE can be mixed together in a wide range of ratios and also form the desired hydrogel.
  • DMAA: SMA: PAE is mixed in a molar ratio of 40: 10: 0.4.
  • the hydrogel precursor solution can be prepared by mixing DMAA, SMA and PAE into water.
  • the pH range of the hydrogel precursor solution is preferably 6-7, e.g., about 6.5.
  • the hydrogel precursor solution can be prepared fresh prior to use, or prepared as a stock solution and diluted prior to use.
  • polymerization activators and/or accelerators such as but not limited to, ammonium persulfate, potassium persulfate, TEMED, VA-044, can be added to the hydrogel precursor solution to induce polymerization or gelation.
  • a biological sample is perfused with a solution comprising a chemical anchoring agent, hydrogel precursors, and a polymerization activator/promoter as described above, i.e., the anchoring and gelation process are performed simultaneously.
  • the sample to be gelated is treated with an anchoring agent prior to contacting with the hydrogel precursor solution, i.e. anchoring followed by gelation.
  • amino acids are connected into peptide chains with a certain sequence through peptide bonds and disulfide bonds.
  • Hydrogen bonds can be formed between amino groups and acyl groups in the polypeptide chain or between peptide chains, making the backbone of the polypeptide chain have a certain regular conformation, including ⁇ -helix, ⁇ -sheet, ⁇ -turn and ⁇ -loop, etc., which is further coiled and folded on the basis of the secondary structure to form a complete spatial conformation.
  • the spatial structure is formed by the non-covalent aggregation of multiple polypeptide chains.
  • protein denaturation treatment also referred to as “homogenization” herein
  • homogenization is performed on the sample-hydrogel composite formed by the gelation reaction, so as to destroy the spatial structure of the protein to facilitate subsequent expansion.
  • the spatial structure of natural-occurring proteins is maintained by secondary bonds such as hydrogen bonds, and the secondary bonds are destroyed after denaturation, and the protein molecule changes from the original ordered coiled compact structure to a disordered loose stretched structure (without changing the primary structure) , thus transforming the protein into a primary structure so that when the sample-hydrogel composite expands, the protein expands with it.
  • denaturation or homogenization is performed by treating samples with SDS based buffer to mechanically homogenize the tissue-hydrogel composite by removing protein-protein interactions due to denaturation.
  • the sample-hydrogel composite is treated with surfactants such as sodium dodecyl sulfate (SDS) or sodium dodecylbenzene sulfonate (SDBS) , etc. to perform protein denaturation.
  • surfactants such as sodium dodecyl sulfate (SDS) or sodium dodecylbenzene sulfonate (SDBS) , etc.
  • the present invention also encompasses a de-anchoring treatment after denaturing the proteins, mainly by breaking/cleaving the amide bond formed by the anchoring between the proteins and the hydrogel network, thereby de-anchoring the proteins from the sample-hydrogel composite.
  • De-anchoring can be performed by increasing the pH of the solution to basic (e.g. by adding a Tris solution) and cleaving the amide bond.
  • sample-hydrogel composite After forming the sample-hydrogel composite, the proteins are denatured and de-anchored, followed by protein staining (such as Coomassie blue) to visualize protein molecules in the sample. A solvent or liquid is added to the sample-hydrogel composite, which is then absorbed by the composite and causes it to expand. Aqueous solutions can be used when the sample-hydrogel composite is water-expandable. Since the polymer embeds the entire tissue sample, as the polymer expands (grows) it also expands the sample. Therefore, the tissue sample itself becomes larger. For example, after incubation in pure water or aqueous buffer, the composite can expand approximately 10-fold in volume, greatly improving the ability of micro-sampling for protein identification. Different sample expansion factors can be obtained by adjusting the incubation time, osmolality of aqueous buffer, temperature, etc., as desired.
  • the molecular chains of the expandable polymer formed throughout the sample expand and separate the biomolecules, allowing the tissue sample itself to become larger. Importantly, the relative positions of the biomolecules remained unchanged after expansion. As the material expands isotropically, the stained labels of the proteins maintain their relative spatial relationship. Once expanded, the tissue can be probed for the presence and/or location of one or more proteins. As shown in the examples, a visualized tissue sample with a linear expansion of about 6 times was obtained, and a microscopic tissue sample with an original diameter of 500 microns could be obtained by using a punch with a millimeter diameter, thereby greatly expanding the accuracy of tissue sampling.
  • the methods disclosed herein may include various combinations of anchoring, gelating, and staining processes.
  • the addition of pure water or other aqueous buffer allows the sample-hydrogel composite to expand up to about 5-8 times in the linear dimension from the initial size of the sample while maintaining high mechanical stability and elasticity. Both mechanical stability and elasticity allow the expanded sample to be easily handled without mechanically deforming the sample hydrogel composite and maintain its integrity.
  • protein identification is performed using high performance liquid chromatography-mass spectrometry (HPLC-MS) .
  • HPLC-MS high performance liquid chromatography-mass spectrometry
  • LC liquid chromatography
  • MS mass spectrometry
  • HPLC-MS is a method routinely used in the art to analyze and measure large molecular weight compounds, such as proteins and polymers.
  • kits for practicing the methods as described herein.
  • the kits may comprise in one or more containers the precursors and/or anchoring agents for forming hydrogels as described above, and optionally, protein disulfide bond reducing agents, protein denaturing agents, staining solutions, various buffers, etc.
  • the precursors are DMAA, SMA and PAE/TPT.
  • the precursors may be present in separate containers, e.g. DMAA in a first container (e.g. in solid or solution form) , SMA in a second container (e.g. in solid or solution form) , and PAE/TPT in a third container (e.g. in solid or solution form) , and mixed and reconstituted before use.
  • any of the following forms can also be adopted: DMAA and SMA are mixed in a suitable ratio and placed in the first container, while PAE/TPT is present in the second container; DMAA and PAE/TPT are mixed in a suitable ratio and placed in the first container, and SMA in the second container; SMA and PAE/TPT are mixed in a suitable ratio and placed in the first container, and DMAA is present in the second container.
  • These containers can be in a combined configuration, or completely separate.
  • the kit may comprise a container in which a mixture of DMAA, SMA and PAE/TPT is present, wherein the molar ratio of DMAA to SMA is in the range of 30: 1 to 3: 1, e.g., in the range of 20: 1 to 3: 1, in the range of 15: 1 to 3: 1, in the range of 10: 1 to 3: 1, in the range of 9: 1 to 3: 1, in the range of 8: 1 to 3: 1, in the range of 7: 1 to 3: 1, in the range of 6: 1 to 3: 1, in the range of 5: 1 to 3: 1, in the range of 4: 1 to 3: 1, more specifically, the molar ratio of DMAA: SMA may be about 4: 1 or 3: 1; and, wherein the molar ratio of SMA: PAE/TPT is in the range of 10: 0.005 to 10: 3, such as 10: 0.05 to 10: 3, 10: 0.08 to 10: 3, 10: 0.1 to 10: 3, 10: 0.2 to 10
  • the precursors for forming the hydrogel are DMAA, SMA and PAE/TPT, and the molar ratio of DMAA: SMA: PAE/TPT is in the range of (40 ⁇ 20) : (10 ⁇ 5) : (0.005-2) , Such as about (40 ⁇ 15) : (10 ⁇ 3.5) : (0.4 ⁇ 0.2) , about (40 ⁇ 10) : (10 ⁇ 2.5) : (0.4 ⁇ 0.2) , about (40 ⁇ 5) : (10 ⁇ 2) : (0.4 ⁇ 0.2) , about (40 ⁇ 5) : (10 ⁇ 1.2) : (0.4 ⁇ 0.2) , about (40 ⁇ 5) : (10 ⁇ 1) : (0.4 ⁇ 0.1) .
  • the precursors for forming the hydrogel are DMAA, SMA, and PAE/TPT, wherein, on a mole basis, if DMAA is 30-50 parts, SMA is 5-15 parts and PAE/TPT is 0.005-1.5 parts.
  • the molar ratio of DMAA: SMA: PAE/TPT can be about 30: 10: 1, 40: 10: 0.08, 40: 10: 0.4, 40: 10: 1, 40: 10: 1.2, or 40: 10: 1.5.
  • the kit may also comprise a container with an anchoring agent, which may be NAS or NSA.
  • the kit may also comprise a container with a polymerization activator or accelerator to add to hydrogel precursor solution prior to polymerization.
  • the polymerization activator or accelerator may be selected from, for example, VA-044, TEMED, potassium persulfate and APS.
  • the kit may also comprise one or more of the following: a container comprising a disulfide bond reducing agent (e.g., TCEP) ; a container comprising a fixative (e.g., paraformaldehyde) ; a container comprising a protein denaturation buffer (e.g., comprising SDS) ; a container containing a protein staining solution (e.g., Coomassie brilliant blue staining solution) ; and a container containing an enzyme for digesting proteins in the sample.
  • a container comprising a disulfide bond reducing agent (e.g., TCEP) ; a container comprising a fixative (e.g., paraformaldehyde) ; a container comprising a protein denaturation buffer (e.g., comprising SDS) ; a container containing a protein staining solution (e.g., Coomassie brilliant blue staining solution) ; and a container containing an enzyme
  • a container is understood to mean any structure that can accommodate or surround the components of the hydrogel formulation, anchoring agent, etc. ; exemplary containers include bottles (e.g., plastic or glass bottles) , syringes, vials, sachets, capsules, ampoules, cartridges, etc. Containers can be shielded from visible, ultraviolet, or infrared radiation by using other components (for example, an aluminum foil bag around the vial) or by choosing the material properties of the container itself (for example, an amber glass vial or an opaque plastic bottle) .
  • the kit may also include a mixing device for mixing the precursors together to produce the hydrogel formulation of the present invention.
  • the kit may also include gel manipulation devices, such as soft brushes, forceps, and/or delivery devices (which may or may not include mixing elements) , etc., for infusing the hydrogel solution onto the sample or transferring the gel.
  • the kit includes one or more of the following components: a gelation chamber configured to accommodate the sample prior to gelation, e.g., a dish with a chamber or groove; a gel manipulation tool, e.g., tweezers , soft brush; a puncher; a cooling device, such as ice pack; multiple Eppendorf tubes, spin columns, etc.
  • a gelation chamber configured to accommodate the sample prior to gelation, e.g., a dish with a chamber or groove
  • a gel manipulation tool e.g., tweezers , soft brush
  • a puncher e.g., a puncher
  • a cooling device such as ice pack
  • the kit may also include other components, such as desiccants or other means of maintaining control of the water content in the kit, indicators to indicate the temperature of the kit, etc., which may be required to preserve the kit in good status during shipping and storage.
  • other components such as desiccants or other means of maintaining control of the water content in the kit, indicators to indicate the temperature of the kit, etc., which may be required to preserve the kit in good status during shipping and storage.
  • the kit includes a gelation chamber for forming the sample-hydrogel composite as described above.
  • the chamber is configured to have cavities, holes or grooves for accommadating the sample prior to gelation, e.g. in the form of a dish, e.g. a MatTek dish.
  • the gelation chamber may also include a lid (e.g., a coverslip) that covers the cavity, well, or groove.
  • the kit further includes an expansion chamber configured to expand the sample and accommodate the sample after gelation.
  • the kit may include multiple vessels packaged together for ease of use.
  • the kit will generally include instructions for using the components of the kit to carry out the methods of the present invention.
  • Instructions for carrying out the methods of the present invention are typically recorded on a suitable recording medium.
  • the instructions may be printed on a material such as paper or plastic.
  • the instructions may be present in the kit as a package insert, in the label of the container of the kit or its components.
  • the instructions are stored as electronically stored data files on a suitable computer-readable storage medium, e.g., the actual instructions are not present in the kit, but are retrieved from a remote source (e.g., a compact disc) .
  • An example of the embodiment is a kit that includes a website where instructions can be viewed and/or downloaded. As with the instructions, the means for obtaining the instructions are documented on suitable materials.
  • the ProteomEx method as disclosed herein provide several advantages including for instance:
  • NSA acrylate-N-succinimidyl ester
  • N, N-Dimethacrylamide (DMAA) , Sodium Methacrylate (SMA) , and Pentaerythritol Allyl Ether (PAE) are formulated in a molar ratio of 40: 10: 0.4, for example, weighing 3.137 g of DMAA, 0.8624 g of SMA, adding 77 ⁇ l of PAE solution, 350 ⁇ l of 10%hydrochloric acid, and 4.6615 ⁇ l of distilled water to prepare a monomer solution, and stored at 4°C.
  • the expansion factor and mechanical strength of hydrogels formed from hydrogel precursor solutions containing different ratios of SMA: DMAA: PAE were tested.
  • the Polymerization initiator used were VA-044 or KPS (potassium persulfate) .
  • the inventors found that the combination of SMA+DMAA+PAE resulted in gels with the desired expansion factor and mechanical strength across a wide range of ratios (Table 1) , indicating that combinations of different ratios of SMA+DMAA+PAE are suitable for successful formulation into a hydrogel.
  • the combination of SMA+DMAA+PAE reached similar effect.
  • the expansion factor is a linear ratio calculated by comparing the sizes (e.g. length and width) of the gelated sample before and after expansion. After adding pure water to swell to the maximum extent, if the gel does not break, it is judged that the mechanical strength of the gel is good. The expansion factor and mechanical strength both as desired was evaluated as achieving a good effect
  • Example 2 Sampling of the expanded sample-hydrogel composite of the brain slice and MS analysis
  • PBS phosphate buffer solution
  • the disulfide bond reduction solution was removed, then the chamber was rinsed three times with 100 mM MES buffer, and incubated with 200 ⁇ l of protein anchoring solution for more than 6 hours at room temperature. The chamber was rinsed three times with 200 ⁇ l of 100 mM MOPS buffer.
  • the chamber was incubated in 200 ⁇ l of activated monomer ATMS solution (activated precursor solution containing APS and TEMED) at 4 °C for more than 6 hours; the solution was removed and a soft brush was used to plate the tissue section onto the bottom of the confocal dish, dried to make the tissue slice closely attached to the bottom of the chamber; then the tissue slice in the chamber was covered with a glass sheet several mms thick, leaving a small gap to add the activated precursor solution again, and incubated at room temperature for about 30 minutes.
  • the covered laser confocal small dish was put into a vacuum drying oven sprinkled with distilled water and preheated to 37°C.
  • a vacuum pump was used to evacuate the vacuum drying oven to a semi-vacuum, and then passing nitrogen to normal atmospheric pressure level, repeated for 4 times.
  • the radical polymerization reaction was conducted for 1.5 hours to fully polymerize the activated precursor solution so as to form a hydrogel.
  • the formed gel was transferred to a 6-well plate, 8 ml of protein denaturation buffer was added, and incubated at 95°C for 3 hours. The gel was then transferred to a 6 cm dish, 10 mL of 50 mM tris buffer pH 8.8 was added, and was eluted at room temperature three times with 0.5 h each time.
  • a 3 mm hole puncher was used and tiny areas of target tissue were selected for punching. The positions and numberings in the order shown in Figure 2 were recorded. Each piece of tissue was placed separately in a 1.5 ml EP tube and corresponding brain region in the brain map was recorded. After sampling, the samples were placed at -4°C for subsequent manipulations.
  • Trypsin was diluted to a final concentration of 12.5 ng/ ⁇ l by adding 25 mM ammonium bicarbonate (ABB) .
  • ABB ammonium bicarbonate
  • the trypsin solution was added to each tube and incubated at 37°C for 4 hours, then the trypsin solution was added again to each tube and incubated at 37°C for 12 hours.
  • Peptide drying The vacuum pump was preheated for 20 minutes, and final drying was performed at 40°C, 6-7 mbar for 4 hours, then stored at -20°C.
  • Peptide solubilization Peptides were solubilized with 30 ul of 2%ACN/0.1%TFA.
  • Micro Spin columns were placed in a new long EP tube, and washed with 20 ⁇ l MeOH by centrifugation, then placed in a new long EP tube, centrifuged and washd with 20 ⁇ l 80%ACN 0.1%TFA, equilibrated with 20 ⁇ l 2%ACN, loaded with 30 ⁇ l of peptide solvent, and centrifuged. Washed with 20 ⁇ l 2%ACN again and centrifuged.
  • Buffer B 100%ACN, 0.1%FA
  • Mouse brain slice samples were collected, added with Tris HCl pH 10, and conducted hydrolysis reaction at 95°C for 30 minutes. Then a lysis solution was added, and lysis was conducted under ultrasonic conditions. A reducing reagent was added to the cleaved protein first, and then incubated at room temperature for 30 minutes in dark, after which an alkylating reagent was added and reacted at 25°C for 30 minutes. Next, trypsin was added to carry out the digestion reaction for a total of 16 hours. At the end of the digestion, 0.1%trifluoroacetic acid was added to terminate the digestion and desalted. The obtained peptide samples were subjected to HPLC-MS/MS for data-dependent acquisition (DDA) . Finally, the proteomic data was de-spectrified by MSFragger software and the number of identified proteins was analyzed.
  • DDA data-dependent acquisition
  • PCT pressure cycle technology
  • 90 cycles of hydrolysis under the cycle program of 50 s, 45,000 psi and 10 s atmospheric pressure were performed.
  • the hydrolyzed proteins were added with reducing and alkylating reagents for reaction.
  • lysC and trypsin were added for PCT-assisted digestion, and the conditions were set to 120 cycles, each cycle consisting of 50 s, 20, 000 psi and 10 s normal pressure.
  • 0.1%trifluoroacetic acid was added to terminate the digestion and desalted.
  • the obtained peptide samples were subjected to HPLC-MS/MS for data-dependent acquisition (DDA) .
  • DDA data-dependent acquisition
  • Table 2 shows the yield of peptides per mg of brain slice samples after expansion gel digestion, in-solution digestion, or pressure cycling digestion, respectively.
  • the yield of peptides is the amount of peptides extracted per mg of tissue. From the results, it can be found that the peptide yield of 48.36 ⁇ g/mg obtained by expansion gel digestion is much higher than that of traditional in-solution digestion (26.01 ⁇ g/mg) , and is also higher than the efficiency of pressure cycling digestion (32.31 ⁇ g/mg) .
  • This method demonstrates the high efficiency of expansion gel enzymatic digestion and can effectively extract peptides for subsequent proteomic analysis.
  • Figure 4 shows the number of polypeptides obtained by sampling the expanded hydrogel with a 3mm puncher and the number of identified protein species.
  • Figure 5 shows the corresponding number of identified protein species ( Figure 5A) and the proportion of missing cleavage after three identical brain slice samples were subjected to expansion gel digestion, in-solution digestion or pressure cycling digestion (ie, PCT-assisted digestion) (Fig. 5B) .
  • the missing cleavage percentage is the percentage of the number of missing cleaved peptides to the total number of peptides identified by the software.
  • the results of the comparison of the three methods show that the expansion gel digestion can obtain comparable identification results in the number of protein species to the other two methods, and has the lowest missing cleavage percentage.
  • the standard deviation of the results obtained by expansion gel digestion was smaller and the stability was better (Fig. 5A) . Meanwhile, the proportion of missing cleavage was significantly reduced, and the efficiency of trypsin digestion was increased (Fig. 5B) .
  • ProteomEx involves chemical treatment and expansion of tissue, it is important to quantify the efficiency of peptide extraction and validate the qualitative and quantitative reproducibility and sensitivity achievable with this method.
  • PCT pressure cycling technology
  • Tissue expansion for proExM-MS was performed according to the following protocol: First, PFA-fixed mouse brain tissue was treated with succinimidyl ester of 6- ( (acryloyl) amino) hexanoic acid (AcX) , 0.1 mg/mL in PBS overnight in a humid chamber at 22°C.
  • Freshly prepared monomer solution (8.6% (w/v) sodium acrylate, 30% (v/v) acrylamide/bisacrylamide (30%solution; 37.5: 1) , 2 M NaCl, 0.01% (w/v) 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-1-oxyl (4-hydroxy TEMPO) inhibitor dissolved in 1x PBS and supplemented with 0.2% (w/v) TEMED, and 0.2% (w/v) APS) was deposited on the tissue slice and evenly spread inside a gelation chamber then covered with glass slide and incubated for 2h at 4°C. Polymerization was carried out in vacuum oven (DZF-6000, Shanghai Sunrise Instrument) at 37°C for 2 h.
  • DZF-6000 Shanghai Sunrise Instrument
  • ProteomEx was characterized by a lower number of missed cleavages (20.4 ⁇ 1.0%) compared to 27.96 ⁇ 1.4%and 24.07 ⁇ 1.2%for in-solution and PCT protocols, respectively, although similar to that for proExM-MS (21.1 ⁇ 5.4%; Figure 6B) .
  • the higher efficiency of peptide digestion and extraction achieved with ProteomEx and proExM-MS can be probably explained by molecular decrowding in the expanded state providing better access for enzyme molecules to the proteolytic sites.
  • Staining of the expanded tissue with the colorimetric dye facilitated visualization of fine morphological features with the naked eye and precise targeting of a region of interest by manual microdissection (down to ⁇ 100 ⁇ m of real size) .
  • a biopsy punch which provided highly reproducible microsampling, to excise 3 mm-diameter tissue-hydrogel composite pieces (corresponding to ⁇ 500 ⁇ m in diameter or 5.9 nL tissue volume before expansion) and adjacent blank hydrogel pieces (used as a control of possible peptide diffusion outside of tissue) from the same expanded mouse brain tissue slice and analyzed as described above using pure MS buffer as a negative control.
  • peptides can be efficiently extracted from the small pieces of the expanded tissue with neglectable diffusion into the blank hydrogel around the tissue. Additionally, compared with whole-brain slices processed by ProteomEx, 3 mm gels identified a slightly lower number of peptides while a comparable number of proteins.
  • PCT-assisted sample preparation as a representative method for processing small samples, enables effective analysis of tissues volume in the range of 0.2-1 ⁇ L, which is about three orders of magnitude higher than ProteomEx.
  • ProteomEx showed a higher degree of reproducibility in the processing of small sample volumes compared to PCT ( Figure 6G, H) .
  • ProteomEx provides a new strategy for sub-nanoliter volume sample preparation for proteomic analysis.
  • Example 4 ProteomEx can be applied to multiple biological tissues with expansion artifacts.
  • ProteomEx To assess the applicability of ProteomEx to various mammalian tissues, we performed ProteomEx on three different mouse tissue types including brain, liver, and breast cancer ( Figure 9A) . Since ProteomEx utilized novel hydrogel composition and optimized homogenization treatment, we first quantified the isotropy of hydrogel-based tissue expansion using a non-rigid registration as done previously for the original protein-retention ExM method (Tillberg et al., Nature Biotechnology, 2016) . The isotropic expansion is essential for precise mapping of spatial proteome distribution onto pre-expanded tissue morphology.
  • proteomic profile with cellular and subcellular features visualized via immunohistochemistry and small dye staining
  • ProteomEx For the 2.52 nL volume of the immunostained tissue, we identified 7000 peptides corresponding to 2000 proteins for three replicates demonstrating the compatibility of ProteomEx with immunohistochemistry.
  • ProteomEx can be readily applied to different mammalian tissue types and is compatible with antibody-stained samples.

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Abstract

A method of physical expanding tissue samples through hydrogel expansion to perform proteomics analysis, and a method for producing expandable sample-hydrogel composites. A hydrogel formulation, an anchoring agent (s) etc. for preparing sample-hydrogel composites, and related kits containing the same.

Description

Methods and Formulations for Expansion Proteomic Analysis of Biological Samples
Cross-References
The present application claimed the benefit of China application No. 202110994923.3, filed on August 27, 2021, incorporated herein entirely be reference.
Technical Field
The present disclosure relates to proteomics, especially a method of proteomics analysis of tissue samples in combination with hydrogel expansion technology, specific formulations used in such methods and uses thereof, as well as kits and systems used for such methods.
Background
Protein subcellular positioning is closely related to the functions of proteins, but traditional high-throughput research methods such as transcriptome sequencing cannot be used for such studies. With the development of microscopy, mass spectrometry and machine learning methods, the study of protein subcellular positioning and its dynamic changes through spatial proteomics is becoming an important tool for understanding cell biology. One of the spatial proteomics research methods is to analyze protein positioning based on imaging without the need to perform cell lysis or isolation of cellular compartments or organelles before proteomics analysis.
Protein imaging is one of the interested areas in the development of imaging mass spectrometry (IMS) technology. IMS directly scans biological samples through mass spectrometry, and analyzes the structural, spatial and temporal distribution information of molecules in cells or tissue. MALDI (Matrix Assisted Laser Desorption Ionization) MS technology developed based on this is capable of multiplex analyzing thousands of analytes on the surface of the samples, to obtain a two-dimensional molecular map that reveals the positions and relative abundance of the molecules, especially allowing the visualization of protein and various protein forms. However, MALDI has limited use due to its relatively low accuracy.
On the other hand, the main solution adopted by the spatial proteomics research of biological tissue samples is to use laser capture microdissection (LCM) methods to obtain tiny tissue samples. LCM can extract a single cell sample from a heterogeneous environment via a microscope combined with a laser aiming system, use lasers to perforate the tissue and remove target area, and separate it from adjacent tissue areas. The polypeptide fragment is obtained through laser sampling and traditional in-solution trypsin digestion, and then rendered for proteomics analysis. However, LCM  sampling can only obtain samples of about 3-5 mm, and the price of LCM is very expensive and difficult for large scale application; traditional in-solution trypsin digestion for treating the obtained samples would lose a lot of low-abundance proteins during the treatment.
There is a need to improve the sampling and analysis method of the spatial proteomics identification, so as to more accurately obtain samples at a specific location and obtain more effective protein fragments in the process of protein treatment.
Summary
The present disclosure solves the problem of effectively obtaining tiny tissue samples, and achieves the analysis of micro-spatial proteomics, the technology that we refer to herein as ProteomEx. Specifically, the present disclosure provides a method of reversibly anchoring a biological sample to a hydrogel and uniformly expanding the formed sample-hydrogel composite. After the biological sample-hydrogel composite are stained and expanded, a visualized tissue sample linearly expanded by about 6 folds can be obtained. Subsequently, tiny tissue samples with original diameter of about 500 microns can be obtained by using a puncher and used for proteomics identification.
In one aspect, the present disclosure provides a method for proteomic identification of a biological sample, comprising:
treating the biological sample with an anchoring agent, adding a hydrogel precursor solution and polymerize, thus forming a sample-hydrogel composite;
performing protein denaturation on the sample-hydrogel composite;
performing protein staining on the sample-hydrogel composite, and let the composite expand; and
sampling from the expanded sample-hydrogel composite for protein identification.
In certain embodiments, the biological sample is selected from a specimen, a tissue slice, a biopsy sample, an organ or part thereof, and an entire organism. The sample can be live, fixed or preserved, such as a fixed tissue slice, a fresh frozen tissue or a tissue slice embedded in paraffin.
In certain embodiments, the sample to be expanded is a tissue slice with a thickness of 50 μm or less, i.e. a thin tissue sample. In some other embodiments, the sample to be expanded is a tissue sample with a thickness of more than 400 μm. In some other embodiments, the sample to be expanded is an entire organ.
In certain embodiments, the method comprises immunostaining (such as antibody staining) of the sample before treated with the anchoring agent. In certain embodiments, immunostaining is performed simultaneously with anchoring.
In some embodiments, the anchoring agent comprises a biomolecule reactive chemical group and a hydrogel reactive chemical group. For example, the anchoring agent can be selected from acrylate-N-succinimidyl ester (NSA) , N- (allyloxycarbonyloxy) -succinimide (NAS) and combinations thereof.
In some embodiments, the anchoring includes contacting the biological sample with an anchor agent, then infusing the biological sample with a hydrogel precursor solution or embedding the biological sample in the hydrogel precursor solution. Alternatively, the anchoring includes infusing the biological sample with a hydrogel precursor solution added with the anchoring agent or embedding the biological sample in the hydrogel precursor solution added with the anchoring agent, thus anchoring and polymerization are performed simultaneously.
The hydrogel precursor solution comprises a precursor (s) of the hydrogel polymer. For example, the precursor (s) is/are selected from sodium acrylate (SA) , sodium methacrylate (SMA) , itaconic acid (IA) , trans-aconitic acid (TAA) , ethyl-2- (hydroxymethyl) -acrylate (EHA) , N, N-methylenebisacrylamide (MBAA, also known as BIS) , N, N-dimethylacrylamide (DMAA) , pentaerythritol tetraacrylate (PT) , propoxylated trimethylol propane triacrylate (TPT) , pentaerythritol triacrylate (PA) , dipentaerythritol pentaacrylate or dipentaerythritol hexaacrylate (DPHA) , trimethylolpropane triacrylate (TTA) , bis (trimethylol) propane) -tetraacrylate (DiTA) , trimethylolpropane trimethacrylate (TTMA) , glycerol propoxylated (1PO/OH) triacrylate (GPT) , ethoxylated trimethylolpropane Triacrylate (TET) , pentaerythritol allyl ether (PAE) , sodium 4-hydroxy-2-methylenebutyrate (SHMB) , N, N-dimethylaminopropylacrylamide (DMPAA) and acrylamide (AA) .
In some embodiments, the precursors comprise SMA, DMAA and PAE. In certain embodiments, the molar ratio of DMAA: SMA included in the precursors is in the range of 30: 1 to 3: 1, e.g., in the range of 20: 1 to 3: 1, in the range of 15: 1 to 3: 1, in the range of 10: 1 to 3: 1, in the range of 9: 1 to 3: 1, in the range of 8: 1 to 3: 1, in the range of 7: 1 to 3: 1, in the range of 6: 1 to 3: 1, in the range of 5: 1 to 3: 1, in the range of 4: 1 to 3: 1, or any sub-range thereof, more specifically, the molar ratio of DMAA: SMA may be about 4: 1 or 3: 1; and wherein the molar ratio of SMA: PAE is in the range of 10: 0.005 to 10: 3, such as 10: 0.008 to 10: 3, 10: 0.01 to 10: 3, 10: 0.02 to 10: 3, 10: 0.03 to 10: 3, 10: 0.04 to 10: 3, 10: 0.05 to 10: 3, 10: 0.06 to 10: 3, 10: 0.07 to 10: 3, 10: 0.08 to 10: 3, 10: 0.09 to 10: 3, 10: 0.1 to 10: 3, 10: 0.4 to 10: 3, 10: 0.6 to 10: 3, 10: 0.8 to 10: 3, 10: 1 to 10: 3, 10: 1.1 to 10: 3, 10: 1.2 to 10: 3, 10: 1.3 to 10: 3, 10: 1.4 to 10: 3, 10: 1.5 to 10: 3, 10: 2 to 10: 3, or any subrange thereof, more specifically, The molar ratio of SMA: PAE can be about 10: 0.005, 10: 0.008, 10: 0.1, 10: 0.5, 10: 1, 10: 1.1, 10: 1.2, 10: 1.3, 10: 1.4, 10: 1.5, 10: 1.6, 10: 1.7, or 10: 1.8.
Further, in certain embodiments, the molar ratio of DMAA: SMA: PAE contained in the precursors is in the range of (40±20) : (10±5) : (0.005-2) , such as about (40±15) : (10±3.5) : (0.005-2) , about (40±10) : (10±2.5) : (0.005-2) , about (40±5) : (10±2) : (0.005-2) , about (40±5) : (10±1.2) : (0.005-2) , about (40±5) : (10±1) : (0.005-2) , preferably about 40: 10: (0.005-2) , about 40: 10: (0.006-2) , about 40: 10: (0.008-2) , about 40: 10: (0.008-1.8) , about 40: 10: (0.05-1.8) , about 40: 10: (0.05-1.6) , about 40: 10: (0.05-1.4) , about 40: 10: (0.08-1.4) , including any sub-ranges thereof. In some other embodiments, DMAA, SMA and PAE are included in the precursor, wherein in molar parts, if DMAA is 30-50 parts, SMA is 5-15 parts, and PAE is 0.005-2 parts.
In some embodiments, the precursors comprise SMA, DMAA and TPT. In certain embodiments, the molar ratio of DMAA: SMA included in the precursors is in the range of 30: 1 to 3: 1, e.g., in the range of 20: 1 to 3: 1, in the range of 15: 1 to 3: 1, in the range of 10: 1 to 3: 1, in the range of 9: 1 to 3: 1, in the range of 8: 1 to 3: 1, in the range of 7: 1 to 3: 1, in the range of 6: 1 to 3: 1, in the range of 5: 1 to 3: 1, in the range of 4: 1 to 3: 1, or any sub-range thereof, more specifically, the molar ratio of DMAA: SMA may be about 4: 1 or 3: 1; and wherein the molar ratio of SMA: TPT is in the range of 10: 0.005 to 10: 3, such as 10: 0.008 to 10: 3, 10: 0.01 to 10: 3, 10: 0.02 to 10: 3, 10: 0.03 to 10: 3, 10: 0.04 to 10: 3, 10: 0.05 to 10: 3, 10: 0.06 to 10: 3, 10: 0.07 to 10: 3, 10: 0.08 to 10: 3, 10: 0.09 to 10: 3, 10: 0.1 to 10: 3, 10: 0.4 to 10: 3, 10: 0.6 to 10: 3, 10: 0.8 to 10: 3, 10: 1 to 10: 3, 10: 1.1 to 10: 3, 10: 1.2 to 10: 3, 10: 1.3 to 10: 3, 10: 1.4 to 10: 3, 10: 1.5 to 10: 3, 10: 2 to 10: 3, or any subrange thereof, more specifically, The molar ratio of SMA: TPT can be about 10: 0.005, 10: 0.008, 10: 0.1, 10: 0.5, 10: 1, 10: 1.1, 10: 1.2, 10: 1.3, 10: 1.4, 10: 1.5, 10: 1.6, 10: 1.7, or 10: 1.8.
Further, in certain embodiments, the molar ratio of DMAA: SMA: TPT contained in the precursors is in the range of (40±20) : (10±5) : (0.005-2) , such as about (40±15) : (10±3.5) : (0.005-2) , about (40±10) : (10±2.5) : (0.005-2) , about (40±5) : (10±2) : (0.005-2) , about (40±5) : (10±1.2) : (0.005-2) , about (40±5) : (10±1) : (0.005-2) , preferably about 40: 10: (0.005-2) , about 40: 10: (0.006-2) , about 40: 10: (0.008-2) , about 40: 10: (0.008-1.8) , about 40: 10: (0.05-1.8) , about 40: 10: (0.05-1.6) , about 40: 10: (0.05-1.4) , about 40: 10: (0.08-1.4) , including any sub-ranges thereof. In some other embodiments, DMAA, SMA and TPT are included in the precursor, wherein in molar parts, if DMAA is 30-50 parts, SMA is 5-15 parts, and TPT is 0.005-2 parts.
In some embodiments, the hydrogel precursor solution also comprises a polymerization activation agent and/or polymerization promoting agent (preferably added before use) , such as VA-044, V50, ammonium persulfate, potassium persulfate and TEMED.
In some embodiments, the sample-hydrogel composite is homogenized by denaturation, preferably a mild denaturation. In some embodiments, the protein degeneration is performed by treating with a detergent (such as SDS or sodium dodecyl benzene sulfonate) .
In some embodiments, the method comprises treating the biological sample with a disulfide bond reductant, and the disulfide bond reductant is e.g. selected from TCEP, DTT, and BME. In some embodiments, the disulfide bond reduction may be performed before anchoring.
In some embodiments, the method further comprises de-anchoring before protein staning. For example, de-anchoring may be performed by increasing pH value to alkaline (for example, by contacting Tris buffer) to break the amide bond.
In some embodiments, the staining is Coomassie blue staining.
In certain embodiments, the expansion of the sample-hydrogel composite is performed by incubating the composite with water or an aqueous buffer. The linear extension factor of the expanded sample-hydrogel composite may be 2-7, for example above 5.
In some embodiments, the sampling is local sampling, such as micro-dissection or local sampling via a punch. The diameter of the samples obtained from local sampling can be less than 3 mm. Preferably, the diameter of the samples obtained by micro-dissection may reach micrometer grade.
In some embodiments, protein identification is performed by mass spectrometry analysis, including enzymatic digestion, peptide extraction (optionally demineralization) and mass spectrometry detection on the sample-hydrogel composite. Mass spectrometry detection can be performed via HPLC-MS or MALDI-TOF.
In one aspect, the disclosure provides a hydrogel formulation comprising a combination of DMAA, SMA and PAE, or a combination of DMAA, SMA and TPT.
In some embodiments, in the hydrogel formulation, the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1, the molar ratio of SMA: PAE is within the range of 10: 0.01 to 10: 3, the molar ratio of DMAA: SMA: PAE is in the range of e.g. (40±20) : (10±5) : (0.005-2) , such as about (40±10) : (10±2.5) : (0.005±0.1) , about (40±5) : (10±1.2) : (0.005±0.1) , about (40±5) : (10±1.2) : (0.005±0.05) , about (40±10) : (10±5) : (0.0005±0.1) , preferably about 40: 10: (0.005-2) , about 40: 10: (0.006-2) , about 40: 10: (0.008-2) , about 40: 10: (0.008-1.8) , about 40: 10: (0.005-1.8) , about 40: 10: (0.005-1.6) , about 40: 10: (0.005-1.4) , about 40: 10: (0.008-1.4) , including any sub-ranges therein. Specifically, the molar ratio of DMAA: SMA: PAE can be about 30: 10: 1, 40: 10: 0.008, 40: 10: 0.4, 40: 10: 1, 40: 10: 1.2, or 40: 10: 1.5.
In some embodiments, in the hydrogel formulation, the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1, the molar ratio of SMA: TPT is within the range of 10: 0.01 to 10: 3, the molar ratio of DMAA: SMA: TPT is in the range of e.g. (40±20) : (10±5) : (0.005-2) , such as about (40±10) : (10±2.5) : (0.005±0.1) , about (40±5) : (10±1.2) : (0.005±0.1) , about (40±5) : (10±1.2) : (0.005±0.05) , about (40±10) : (10±5) : (0.0005±0.1) , preferably about 40: 10: (0.005- 2) , about 40: 10: (0.006-2) , about 40: 10: (0.008-2) , about 40: 10: (0.008-1.8) , about 40: 10: (0.005-1.8) , about 40: 10: (0.005-1.6) , about 40: 10: (0.005-1.4) , about 40: 10: (0.008-1.4) , including any sub-ranges therein. Specifically, the molar ratio of DMAA: SMA: TPT can be about 30: 10: 1, 40: 10: 0.008, 40: 10: 0.4, 40: 10: 1, 40: 10: 1.2, or 40: 10: 1.5.
In some embodiments, the hydrogel formulation is prepared as a stock solution of DMAA, SMA, and PAE diluted in water, or DMAA, SMA and TPT diluted in water. Alternatively, the hydrogel formulation is prepared as a mixture of DMAA, SMA, and PAE without dilution, or DMAA, SMA and TPT without dilution.
In one aspect, the disclosure provides use of the hydrogel formulation as disclosed herein for expansion of a biological sample, wherein the hydrogel formulation comprises hydrogel precursors comprising DMAA, SMA and PAE, or DMAA, SMA and TPT.
In one aspect, the disclosure provides a kit, comprising DMAA, SMA and PAE in one or more containers, or DMAA, SMA and TPT in one or more containers.
In some embodiments, DMAA, SMA, and PAE are each in separated containers, respectively. Optionally, the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1, the molar ratio of SMA: PAE is within the range of 10: 0.005 to 10: 3. Alternatively, at least two of DMAA, SMA, and PAE are mixed in one container. The ratios of DMAA, SMA, and PAE may be as described as above.
In some embodiments, DMAA, SMA, and TPT are each in separated containers, respectively. Optionally, the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1, the molar ratio of SMA: TPT is within the range of 10: 0.005 to 10: 3. Alternatively, at least two of DMAA, SMA, and TPT are mixed in one container. The ratios of DMAA, SMA, and TPT may be as described as above.
In some embodiments, the kit is suitable for storage at 0℃-4℃ or at room temperature.
In some embodiments, the kit further contains one or more containers comprising NAS and/or NSA as anchor agents.
In some embodiments, the kit further contains one or more of the following:
a container comprising a disulfide bond reductant (such as TCEP) ; a container comprising a polymerization activation agent or polymerization promoting agent selected from VA-044, V50, TEMED, ammonium persulfate and potassium persulfate; a container comprising a fixative (such as paraformaldehyde) ; a container comprising a protein denaturation buffer (such as a protein denaturation buffer comprising SDS) ; a container comprising protein staining solution (such as Coomassie blue staining buffer) ; a container comprising enzymes (such as trypsin) for digestion of proteins in the sample; and a container comprising one or more buffers.
In some embodiments, the kit further comprises one or more of the following: a gelation room configured to accommodate the sample before gelation, such as a dish with cavity or grooves; a staining room configured to stain the proteins in the sample; a hydrogel operation tool, such as a tweezer, soft brush; a puncher; and a cooling device, such as ice bags.
This disclosure also provides the kit as described above for physical expansion of biological samples.
In one aspect, the disclosure provides a system of spatial proteomics analysis of a biological sample, including:
In one aspect, provided herein is a system for spatial proteomics analysis of a biological sample, comprising:
an anchoring module for anchoring the proteins in the biological sample;
a gelation module for sample anchoring and hydrogel polymerization to form a sample-hydrogel composite;
a protein degeneration and de-anchoring module for denaturation and de-anchoring of the proteins in the sample-hydrogel composite respectively;
a staining module for protein staining and optionally local sampling of the sample-hydrogel composite; and
optionally, mass spectrometry analysis module for pre-processing of the samples obtained by local sampling before mass spectrometry and which is connected with a mass spectrometry detection device.
In certain embodiments, the system further comprises a protein pre-processing module for disulfide bond reduction of the biological sample, and/or immune-staining.
In certain embodiments, one or more of the modules can be integrated together. For example, the anchoring module and gelation module can be integrated into one module, thereby anchoring and gelation are performed at the same time.
From the detailed description of the following embodiments in connection with the figures, the aforementioned and other characteristics and advantages of this disclosure will become obvious.
Description of Figures
Figure 1 illustrates ProteomEx workflow. (A) Chemically fixed tissue samples, which can be immunostained beforehand, are treated with the chemical anchor, embedded into the hydrogel, and mechanically homogenized by mild denaturation. The Coomassie-stained (CBB) hydrogel embedded samples are expanded and imaged. After imaging, expanded samples are microdissected and excised pieces of the tissue-hydrogel composite are processed by in-gel digestion to recover  peptides for LC-MS/MS analysis (acronyms LEF, linear expansion factor, VOL, volumetric expansion factor) . (B) Timeline of ProteomEx indicating total duration and hands-on time of each step (total duration/hands-on time) . (C) Brightfield images of mouse brain tissue section before expansion and (D) after Coomassie staining and expansion (E) showing automatically detected and annotated brain regions (LEF = 5.5-fold) .
Figure 2 illustrates a schematic diagram of manual local sampling of a brain slice sample-hydrogel composite after expansion using a 3 mm punch, the left panel is the sample before punching, and the right panel is the sample after punching.
Figure 3 illustrates the size of the brain slice sample-hydrogel composite before and after swelling, the left panel is the size of the sample before swelling, the right panel is the size of the sample after swelling. It can be seen that the brain slice swelled about 6.1 times.
Figure 4 illustrates a graph of the number of protein species obtained by proteomics identification after the expanded gel was punched with a 3 mm puncher for sampling.
Figure 5 illustrates a histogram of the number of identified protein species (A) and the proportion of missing cleavage (B) for brain slice samples subjected to swelling gel digestion, in-solution digestion, and pressure cycling digestion (from left to right) .
Figure 6 illustrates ProteomEx benchmarking against standard methods in the field. (A) The peptide yields of the in-solution, PCT, proExM-MS, and ProteomEx methods applied to the mouse brain tissue (n=4, 4, 7, 4 technical replicates from one, one, two, and one brain slices, respectively, the same samples were used to acquire data shown in panels A-F; dot, individual data point, bar, mean, whiskers, standard deviation, throughout Figure 6; Welch's t-test) . (B) Missed cleavages of the identified peptides prepared using in-solution, PCT, proExM-MS, and ProteomEx methods. (C, D) Number of peptide (C) and protein (D) identifications in seven sample groups (n=3 and 3 punches from one slice from one mouse for ProteomEx (5.9 nL) and blank hydrogel, n=3 independent injections for MS buffer; analyzed by DDA) . (E) Venn diagram of identified proteins for the bulk samples shown in D. (F) The subcellular locations of the identified proteins for the samples shown E. (G, H) Number of peptide (G) and protein (H) identifications for different tissue volumes processed by ProteomEx and PCT (n=4 punches per group from 2 mice for ProteomEx, LEF = 6.3, 6.2, 6.3, 5.9; n=3 tissue dissections per group from 1 mouse for PCT; solid line, four-parameter logistic fit, dashed line indicates 95%confidence interval border, shadowed area represents 95%confidence interval; analyzed by PulseDIA) .
Figure 7 illustrates reproducibility and stability comparison for the four methods. (A) Heatmap of Pearson correlations of each paired samples from the four strategies analyzed using the  MSFragger software. The color bar indicates the values of Pearson correlations. (B) Coefficient of variation of quantified protein abundance from the four methods.
Figure 8 illustrates peptide modifications analysis. (A) The upset plot showing the number of overlapped modifications identified in the four methods by open search mode. (B) Distribution of different modifications ranked by peptide modification ratio from ProteomEx. The four color indicate corresponding methods.
Figure 9 illustrates validation of ProteomEx in different tissue types. (A) Representative bright-field images of different tissue slices pre-expansion (left column) and post-expansion (middle column; Coomassie-stained) and overlay (right column) of pre-expansion image (green pseudo-color) and registered post-expansion image (magenta pseudo-color) . Arrows represent the deformation vector field (n=3, 3, and 3 tissue slices from one mouse each; in the images of the expanded sample white circles represent the dissected area for peptide analysis shown in C) . (B) The root-mean-square (RMS) measurement length error for pre-versus post-expansion brain slice images for the experiments shown in A (n=3, 3, and 3 tissue slices from one mouse each; average LEF = 5.8, 6.5, and 6.2 for brain, liver, and tumor respectively) . (C, D) Number of peptide (C) and protein (D) identifications for the (n=3, 3, and 3 punches for each tissue from one mouse each; 5.6 nL tissues were punched out and analyzed by DDA) . (E) Number of peptide and protein identifications for 0.37 nL brain tissue analyzed in PulseDIA mode (n= 3 punches from one slice) .
Detailed Description
The beneficial effects of the present invention are as follows: after anchoring the protein to the hydrogel, stained and uniformly expanded, tissue samples with a diameter of about 100-1000 (e.g. 128-1000) microns can be manually obtained to determine accurate spatial positioning of the proteins in the tissue sample; by anchoring the proteins to the hydrogel, the problem of losing a large amount of proteins in the traditional in-solution trypsin digestion method can be avoided; the protein anchored in the hydrogel can be more easily digested by trypsin after denaturation, lipid removal and swelling, obtaining more effective peptide fragments; tiny tissue samples treated with this protocol can identify more proteins than traditional in-solution trypsin digestion and pressure cycling technology of the second generation. The chemical reagents and processing conditions used in the methods of the present disclosure are more economic and easily available, thus more suitable for large-scale applications and accessible for an average biochemical and analytical laboratories and facilities.
The disclosure and embodiments set forth herein are to be construed as exemplary only, and not as limiting of the scope of the invention. Although specific terms are employed herein, unless  otherwise stated, they are used in a generic and descriptive sense and not for purposes of limitation. All references cited herein, including publications, patents, and patent applications, are incorporated by reference in their entirety.
Definitions
As used herein, the singular forms "a, " "an, " and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.
As used herein, the term "expand" , "expansion" , "swell" , “magnification” , "swelling" and their grammatical variations generally refer to expansion or enlargement, e.g., uniform expansion in 3 dimensions, that occurs upon contact with a liquid such as water or other solvents. The expandable material is preferably transparent so that light can pass through the sample when expanded. As exemplified herein, the swellable material may be a hydrogel. In some embodiments, the expandable material is formed from the polymerization of its precursors, monomers or oligomers. For example, the monomer may contain a water-soluble group containing a polymerizable ethylenically unsaturated group. The monomer or oligomer may comprise one or more of the following: substituted or unsubstituted methacrylates, acrylates, acrylamides, methacrylamides, vinyl alcohols, vinylamines, allylamines, allyl alcohols, including its divinyl crosslinkers (e.g., N, N-alkylene bisacrylamides) . In some embodiments, the expandable material is polyacrylates and copolymers or cross-linked copolymers thereof. Alternatively, the swellable material can be formed in situ by chemically crosslinking water-soluble oligomers or polymers. In the present invention, it is contemplated that a precursor solution of a swellable material is added to the sample, allowing the precursor to polymerize and swell in situ.
As used herein, the term "Expansion proteomics" refers to a process of reversible anchoring of biological samples (usually tissue samples) to hydrogels that can be uniformly swelled, and the formed sample-hydrogel composite is subjected to protein denaturation, de-anchoring, staining, swelling, sampling and enzymatic digestion to obtain polypeptides for proteomic analysis (especially spatial proteomics analysis) .
Expansion Proteomics Analysis of Biological Samples
The present disclosure provides a method for the spatial proteomics analysis of proteins from spatially distinct regions of a biological sample (e.g. a tissue section) . In some embodiments, the method comprises polymerizing a sample with hydrogel precursors to form a sample-hydrogel composite, staining proteins in the sample, swelling the sample-hydrogel composite, and acquiring micro-samples from the expanded composite for protein identification. Further, the protein  identification includes identification of peptides sampled and extracted from the composite by mass spectrometry techniques (eg, LC-MS/MS) .
In one aspect, the present disclosure provides a method of expanding a tissue sample to obtain tiny tissue samples (e.g. at μm grade) , wherein a sample-hydrogel composite is formed in a manner that preserves the relative positions of biomolecules (e.g. proteins) in their original functional status. Since the relative positions of the proteins are preserved, the localization information of the proteins at different positions in the sample can be acquired by sampling different regions of the gel and identifying the proteins.
In some embodiments, the method as disclosed herein comprises one or more of the following steps:
1) Optionally, pre-processing the biological sample (fixed or living) , for example immunostaining or treating the sample with a disulfide bond reductant;
2) Treating the biological sample with an anchoring agent, adding a hydrogel precursor solution and polymerize to form a sample-hydrogel composite, wherein the anchoring and polymerization can occur at the same time or sequentially;
3) Performing protein denaturation and de-anchoring on the sample-hydrogel composite;
4) Optionally, washing the sample-hydrogel composite with an organic solvent such as methanol;
5) Performing protein staining on the sample-hydrogel composite, then let the composite expand; and
6) Sampling from the expanded sample-hydrogel composite for proteomics identification.
The present disclosure also contemplates de-swelling the sample-hydrogel complex, e.g. returning to its original size, by soaking in a high salt buffer. Therefore, the method of the present invention is able to reversibly modulate the expansion factor of the sample-hydrogel composite.
The present disclosure also exemplifies some chemical anchoring agents or combinations thereof that can be used for protein anchoring, which can more rapidly penetrate larger biological samples to anchor biomolecules. The methods herein are applicable not only to cells and thin samples, but also to large or thick samples.
The hydrogel obtained in the methods herein has high mechanical stability after expansion, allowing its application to expand large tissue blocks, tissue slices, whole organs and even whole organisms without deforming and mechanically disintegrating under its own weight, which show obvious advantages compared to other hydrogels. In some embodiments, the hydrogel precursor solution or hydrogel formulation from which the hydrogel is formed comprises DMAA, SMA, and PAE.
The methods of the present disclosure can be used for proteomic (especially spatial proteomic) studies of different organs and tissues, such as brain mapping, connectome studies as well as new diagnostics, personalized medicine, histopathology and other medical applications.
Biological Samples for Hydrogel Expansion
Biological samples for hydrogel anchoring and expansion in the present disclosure can be fresh, frozen, or fixed (i.e. preserved) . The source of biological samples can be a solid tissue, for example from a fresh, frozen and/or preserved organ, or a tissue sample or biopsy or aspiration sample, including preserved tumor samples such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples. A tissue sample may be one that is routinely prepared and preserved in routine clinical practice. Fixed samples are histologically preserved samples using a fixative (e.g., formaldehyde, paraformaldehyde) . Fixation can be accomplished before, during, or after the disulfide bond reduction treatment, protein anchoring, and gelation. In certain embodiments, the sample of interest is fixed prior to disulfide bond reduction treatment. Samples can also be embedded in solid and generally rigid media such as paraffin, wax, collodion or resin, so that thin sections can be cut for processing.
Fixation can be carried out by conventional methods. Those skilled in the art will understand that the choice of fixative will depend on the purpose for which the sample is to be stained histologically or otherwise analyzed. Those skilled in the art will also understand that the time of fixation depends on the size of the tissue sample and the fixative used. For example, neutral buffered formalin, Bouin or paraformaldehyde can be used to fix the sample.
In certain embodiments, the tissue sample is a thin tissue section (as thin as 5 μm) , such as a brain section, liver section, or tumor section. In some other embodiments, the sample used for expansion is a tissue sample with a thickness greater than 400 μm or even a whole organ and parts thereof, the organ including but not limited to brain, spinal cord, heart, lung, liver, kidney, stomach, colon, bone, muscle, skin, lymph nodes, genitals, breast, pancreas, prostate, bladder, thyroid and eyes, etc.
Disulfide Bond Reduction of Biological Samples
A disulfide bond is a chemical bond that connects the sulfhydryl groups of two cysteine residues in different peptide chains of a protein or in the same peptide chain. Disulfide bonds are relatively stable covalent bonds, which play a role in stabilizing the spatial structure of peptide chains in protein molecules. The larger the number of disulfide bonds, the greater the stability of the protein molecule against the influence of external factors. Therefore, in order to promote the  anchoring of proteins and hydrogels in the subsequent process, a disulfide bond reduction treatment may be performed on the sample to destroy the disulfide bonds of protein molecules therein.
Disulfide bonds in the sample can be broken by treatment with reducing agents. There are a variety of reducing agents known in the art to achieve disulfide bond cleavage, commonly used reducing agents are thiols such as β-thioethanol (β-ME) or dithiothreitol (DTT) . Excess thiol reagent is usually used to ensure complete cleavage of the disulfide bonds. Other reducing agents include TCEP. Unlike β-ME and DTT, TCEP is odorless, selective, can work in alkaline and acidic environments (unlike DTT) , is more hydrophilic, and is resistant to oxidation.
In some embodiments of the invention, the biological sample is incubated with a reducing agent for a sufficient period of time for the disulfide bond reduction treatment, wherein the reducing agent is selected from the group consisting of TCEP, β-ME and DTT. The reducing agent can be formulated as a solution, for example, a solution of TCEP can be prepared by dissolving the hydrochloride salt form of TCEP in boric acid buffer.
Anchoring of Biological Samples
To form a swellable sample-hydrogel composite, the sample is contacted with a chemical anchoring agent or perfused with an anchoring agent solution, and is incubated for a sufficient time for the chemical reaction to occur.
The terms "anchoring agent" and "crosslinking agent" are used interchangeably herein. The chemical anchoring agent is preferably a bifunctional anchor comprising a biomolecule reactive chemical group and a hydrogel reactive chemical group. In certain embodiments, chemical anchoring agents containing one or more functional groups attach reactive groups to functional groups (e.g., primary amines or sulfhydryls) of biomolecules within the biological sample, which may be proteins, nucleic acids, lipids, proteoglycans, lipopolysaccharides, etc. Since the present disclosure is focused on proteomics study, the anchoring agents employed are those that are preferred towards anchoring proteins. The anchoring agent functionalizes biomolecules such as proteins in the sample to react with the growing chains of the hydrogel polymer upon polymerization of the hydrogel, thereby covalently anchoring the functionalized biomolecules to the hydrogel network.
Chemical anchoring agents are usually provided in the form of anchoring agent solutions. In some embodiments, the anchoring agent solution is prepared by dissolving or diluting the chemical anchoring agent in an aqueous buffer such as PBS, MES buffer. The anchoring agent solution may also contain a fixing agent to fix the sample at the same time as anchoring. One skilled in the art can  easily determine the buffers for preparing the anchoring agent solution based on the selected anchoring agent.
In certain embodiments, the chemical anchoring agent comprises two distinct functional groups, one for functionalization of biomolecules, particularly protein molecules, and the other for reaction with growing polymer chains. Functionalization of biomolecules can be performed during chemical fixation, i. e., on live or freshly frozen samples, or after chemical fixation, on fixed and preserved samples. As understood by those skilled in the art, the two distinct functional groups of a bifunctional chemical anchor can be separated by a chemical spacer of various lengths and structures, including branched spacers.
In some embodiments, a biomolecule reactive chemical group includes, but not limited to, N-hydroxysuccinimide (NHS) esters, epoxy groups, aldehyde groups, carboxamide groups, which can react with amino or carboxylic acid groups on proteins, peptides, nucleic acids and/or lipids. A hydrogel reactive group includes, but not limited to, vinyl, allyl, acrylate, methacrylate, acrylonitrile, acrylamide groups.
In some preferred embodiments, the chemical anchoring agent used to directly crosslink the protein to any hydrogel polymer chain is selected from the group consisting of acrylate-N-succinimidyl ester (NSA) , N- (allyloxycarbonyloxy) -succinimide (NAS) , and a combination thereof. Treatment with NSA or NAS modifies the amine on the protein with acrylamide or allyloxycarbonyloxyamide functional group, respectively, allowing the functionalized protein to anchor to the hydrogel during polymerization.
In some embodiments, the chemical anchoring agent comprises NSA. In other embodiments, the anchoring agent comprises NAS. In still other embodiments, the anchoring agent comprises both NAS and NSA, e.g., NAS and NSA at a 1: 1 molar ratio. The anchoring agent can be diluted in the anchoring buffer at various ratios, e.g., about 1: 10, 1: 20, 1: 30, 1: 40, 1: 50, 1: 100, 1: 150, 1: 200, depending on the choice of buffer and anchoring agent, desired reaction rate and anchoring effect, among others.
Hydrogel Polymerization/Gelation
As disclosed above, a biological sample is permeated (e.g., infused, injected, impregnated, added, or mixed in other ways) or embedded in a hydrogel precursor solution, and then polymerization takes place to form a swellable sample-hydrogel composite. The composite comprises a swellable polymer network in which protein molecules within the sample (either already functionalized or being functionalized by an anchor) are covalently bound to the polymer chains of the hydrogel.
Hydrogel precursor solutions typically comprise one or more hydrogel monomers, oligomers or precursors dissolved in an aqueous solution such as water or an aqueous solution added with an acid to adjust pH. The terms "monomer" or "precursor" in relation to hydrogel are used interchangeably herein to include monomers, co-monomers, and crosslinkers that build the small units of the hydrogel polymer chain. In addition to hydrogel precursor, the hydrogel precursor solution can also be added with polymerization activators or accelerators such as VA-044, V50, APA, potassium persulfate and TEMED prior to use. Water or an aqueous solution is typically used as the dispersion medium in the forming hydrogel precursor solution, but other solvents can also be used.
The hydrogel precursor will polymerize/cross-link with the sample to form a hydrogel-sample composite. In certain embodiments, the precursors of hydrogel include, but are not limited to, sodium acrylate (SA) , sodium methacrylate (SMA) , itaconic acid (IA) , trans-aconitic acid (TAA) , ethyl-2- (hydroxymethyl) -acrylate (EHA) , N, N-methylenebisacrylamide (MBAA, also known as BIS) , N, N-dimethylacrylamide (DMAA) , pentaerythritol tetraacrylate (PT) , propoxylated trimethylol Propane triacrylate (TPT) , pentaerythritol triacrylate (PA) , dipentaerythritol pentaacrylate or dipentaerythritol hexaacrylate (DPHA) , trimethylolpropane triacrylate (TTA) , bis(trimethylol) Propane) -tetraacrylate (DiTA) , trimethylolpropane trimethacrylate (TTMA) , glycerol propoxylated (1PO/OH) triacrylate (GPT) , ethoxylated trimethylolpropane Triacrylate (TET) , pentaerythritol allyl ether (PAE) , sodium 4-hydroxy-2-methylenebutyrate (SHMB) , N, N-dimethylaminopropylacrylamide (DMPAA) and acrylamide (AA) . All reagents are commercially available, chemically stable and relatively safe (can be used in routine biological laboratories) .
For example, if the sample of interest is to be embedded in a sodium polyacrylate based hydrogel, the sample may be perfused with a solution comprising the monomers sodium acrylate and acrylamide as well as the anchoring agent N, N-methylenebisacrylamide. A polymerization accelerator or initiator is added to the solution prior to infusion, so that upon permeation of the sample the solution is also activated to form the hydrogel polymer. Preferably, such monomer-containing solutions are aqueous.
The hydrogels are expected to maintain high mechanical stability in the expanded state to achieve tunable and reversible expansion. To this end, the precursor of the hydrogel can be selected from N, N-dimethylacrylamide (DMAA) , sodium methacrylate (SMA) and pentaerythritol allyl ether (PAE) , preferably comprising all three. Alternatively, the precursor of the hydrogel can be selected from N, N-dimethylacrylamide (DMAA) , sodium methacrylate (SMA) and propoxylated trimethylol Propane triacrylate (TPT) , preferably comprising all three. In certain embodiments, DMAA, SMA and PAE are selected as precursors, wherein the molar ratio of DMAA to SMA is in the range of 30: 1 to 3: 1, such as in the range of 20: 1 to 3: 1, in the range of 15: 1 to 3: 1, in the range of 10: 1 to 3: 1,  in the range of 9: 1 to 3: 1, in the range of 8: 1 to 3: 1, in the range of 7: 1 to 3: 1, in the range of 6: 1 to 3: 1, in the range of 5: 1 to 3: 1, in the range of 4: 1 to 3: 1, more specifically, the molar ratio of DMAA: SMA may be about 4: 1 or 3: 1; and wherein the molar ratio of SMA: PAE is in the range of 10: 0.005 to 10: 3, e.g., in the range of 10: 0.05 to 10: 3, 10: 0.08 to 10: 3, 10: 0.1 to 10: 3, 10: 0.2 to 10: 3, 10: 0.3 to 10: 3, 10: 0.4 to 10: 3, 10: 0.5 to 10: 3, 10: 0.6 to 10 : 3, 10: 0.7 to 10: 3, 10: 0.8 to 10: 3, 10: 0.9 to 10: 3, 10: 1 to 10: 3, 10: 1.1 to 10: 3, 10: 1.2 to 10: 3 , 10: 1.3 to 10: 3, 10: 1.4 to 10: 3, 10: 1.5 to 10: 3, 10: 2 to 10: 3, more specifically, the molar ratio of SMA: PAE may be about 10 : 0.05, 10: 0.08, 10: 0.1, 10: 0.5, 10: 1, 10: 1.1, 10: 1.2, 10: 1.3, 10: 1.4, 10: 1.5, 10: 1.6, 10: 1.7, or 10: 1.8. In some embodiments, PAE can be replaced by TPT.
Further, in certain embodiments, the precursors comprise DMAA, SMA and PAE, and the molar ratio of DMAA: SMA: PAE is in the range of (40±20) : (10±5) : (0.005-2) , such as about (40±15) : (10±3.5) : (0.4±0.2) , about (40±10) : (10±2.5) : (0.4±0.2) , about (40±5) : (10±2) : (0.4±0.2) , about (40±5) : (10±1.2) : (0.4±0.2) , about (40±5) : (10±1) : (0.4±0.1) . In some other embodiments, the precursor comprises DMAA, SMA, and PAE, wherein, on a mole basis, if DMAA is 20-60 parts, SMA is 5-15 parts, and PAE is 0.005-1.5 parts. Specifically, the molar ratio of DMAA: SMA: PAE can be about 30: 10: 1, 40: 10: 0.008, 40: 10: 0.4, 40: 10: 1, 40: 10: 1.2, or 40: 10: 1.5.
The precursor solution can be formulated by adding water or an aqueous solution (e.g., an aqueous solution with the addition of an acid to adjust the pH) to the hydrogel precursor or by dissolving the hydrogel precursor in water or an aqueous solution. For example, the concentration of the hydrogel precursor contained in the precursor solution may be in the range of 30-60%on a weight basis (w/w) , such as 30%, 40%, 50%or 60%. Stock solutions of hydrogel precursor solutions can be prepared and diluted prior to use. Stock solutions may have high concentrations (w/w) of precursors, e.g., about 50%or higher, about 75%or higher, about 80%or higher, about 90%or higher. The stock solution can be diluted to a concentration of about 30-50%when applied to the sample.
As exemplified in the Examples, using DMAA, SMA and PAE as precursors, the obtained hydrogel has superior mechanical strength and expansion factor properties. The precursors can be mixed in various ratios to form hydrogels that swell adjustably and reversibly in aqueous buffers at an expansion factor of up to 7-fold or more in the linear dimension. The inventors have found that DMAA: SMA: PAE can be mixed together in a wide range of ratios and also form the desired hydrogel.
Specifically, in one embodiment, DMAA: SMA: PAE is mixed in a molar ratio of 40: 10: 0.4. The hydrogel precursor solution can be prepared by mixing DMAA, SMA and PAE into water. The pH range of the hydrogel precursor solution is preferably 6-7, e.g., about 6.5. The hydrogel  precursor solution can be prepared fresh prior to use, or prepared as a stock solution and diluted prior to use.
Before ready for polymerization, polymerization activators and/or accelerators such as but not limited to, ammonium persulfate, potassium persulfate, TEMED, VA-044, can be added to the hydrogel precursor solution to induce polymerization or gelation.
In some embodiments, a biological sample is perfused with a solution comprising a chemical anchoring agent, hydrogel precursors, and a polymerization activator/promoter as described above, i.e., the anchoring and gelation process are performed simultaneously. Alternatively, the sample to be gelated is treated with an anchoring agent prior to contacting with the hydrogel precursor solution, i.e. anchoring followed by gelation.
Protein Denaturation and De-anchoring
In protein molecules, amino acids are connected into peptide chains with a certain sequence through peptide bonds and disulfide bonds. Hydrogen bonds can be formed between amino groups and acyl groups in the polypeptide chain or between peptide chains, making the backbone of the polypeptide chain have a certain regular conformation, including α-helix, β-sheet, β-turn and Ω-loop, etc., which is further coiled and folded on the basis of the secondary structure to form a complete spatial conformation. The spatial structure is formed by the non-covalent aggregation of multiple polypeptide chains.
Preferably, protein denaturation treatment (also referred to as “homogenization” herein) is performed on the sample-hydrogel composite formed by the gelation reaction, so as to destroy the spatial structure of the protein to facilitate subsequent expansion. The spatial structure of natural-occurring proteins is maintained by secondary bonds such as hydrogen bonds, and the secondary bonds are destroyed after denaturation, and the protein molecule changes from the original ordered coiled compact structure to a disordered loose stretched structure (without changing the primary structure) , thus transforming the protein into a primary structure so that when the sample-hydrogel composite expands, the protein expands with it.
In some embodiments, denaturation or homogenization is performed by treating samples with SDS based buffer to mechanically homogenize the tissue-hydrogel composite by removing protein-protein interactions due to denaturation. In some embodiments of the present disclosure, after the gelation reaction, the sample-hydrogel composite is treated with surfactants such as sodium dodecyl sulfate (SDS) or sodium dodecylbenzene sulfonate (SDBS) , etc. to perform protein denaturation.
Further preferably, the present invention also encompasses a de-anchoring treatment after denaturing the proteins, mainly by breaking/cleaving the amide bond formed by the anchoring  between the proteins and the hydrogel network, thereby de-anchoring the proteins from the sample-hydrogel composite. De-anchoring can be performed by increasing the pH of the solution to basic (e.g. by adding a Tris solution) and cleaving the amide bond.
Expansion of the Sample-hydrogel Composite
After forming the sample-hydrogel composite, the proteins are denatured and de-anchored, followed by protein staining (such as Coomassie blue) to visualize protein molecules in the sample. A solvent or liquid is added to the sample-hydrogel composite, which is then absorbed by the composite and causes it to expand. Aqueous solutions can be used when the sample-hydrogel composite is water-expandable. Since the polymer embeds the entire tissue sample, as the polymer expands (grows) it also expands the sample. Therefore, the tissue sample itself becomes larger. For example, after incubation in pure water or aqueous buffer, the composite can expand approximately 10-fold in volume, greatly improving the ability of micro-sampling for protein identification. Different sample expansion factors can be obtained by adjusting the incubation time, osmolality of aqueous buffer, temperature, etc., as desired.
The molecular chains of the expandable polymer formed throughout the sample expand and separate the biomolecules, allowing the tissue sample itself to become larger. Importantly, the relative positions of the biomolecules remained unchanged after expansion. As the material expands isotropically, the stained labels of the proteins maintain their relative spatial relationship. Once expanded, the tissue can be probed for the presence and/or location of one or more proteins. As shown in the examples, a visualized tissue sample with a linear expansion of about 6 times was obtained, and a microscopic tissue sample with an original diameter of 500 microns could be obtained by using a punch with a millimeter diameter, thereby greatly expanding the accuracy of tissue sampling.
In some embodiments, the methods disclosed herein may include various combinations of anchoring, gelating, and staining processes. In certain embodiments, the addition of pure water or other aqueous buffer allows the sample-hydrogel composite to expand up to about 5-8 times in the linear dimension from the initial size of the sample while maintaining high mechanical stability and elasticity. Both mechanical stability and elasticity allow the expanded sample to be easily handled without mechanically deforming the sample hydrogel composite and maintain its integrity.
Mass Spectrometry (MS) Analysis
Traditional approaches to proteomic identification involve one or more of the following steps: microextraction from the tissue surface with aqueous or organic solvents, tissue homogenization of  multiple adjacent tissue regions, laser capture microdissection (LCM) collection of the regions of interest, or high-performance liquid chromatography (HPLC) fractionation, and aliquots of these fractions were subjected to MS analysis to determine which fractions contain the protein (s) of interest.
In some embodiments, protein identification is performed using high performance liquid chromatography-mass spectrometry (HPLC-MS) . Liquid chromatography (LC) can effectively separate the organic components in the samples to be tested, while mass spectrometry (MS) can analyze the separated organics one by one to obtain information on the molecular weight, structure and concentration of the organics. HPLC-MS is a method routinely used in the art to analyze and measure large molecular weight compounds, such as proteins and polymers.
Matching mass spectrometry data to protein databases is crucial to proteomic identification. At present, most of the proteomic studies use trypsin, which can specifically digest the carboxyl terminus of lysine and arginine residues. Despite the high specificity of trypsin cleavage, missing trypsin cleavage is not uncommon in the identification of proteins. Therefore, the pros and cons of different biological sample processing conditions can be evaluated by analyzing the percentages of missing cleavage.
Kits
The present disclosure also provides kits for practicing the methods as described herein. The kits may comprise in one or more containers the precursors and/or anchoring agents for forming hydrogels as described above, and optionally, protein disulfide bond reducing agents, protein denaturing agents, staining solutions, various buffers, etc. Preferably, the precursors are DMAA, SMA and PAE/TPT. The precursors may be present in separate containers, e.g. DMAA in a first container (e.g. in solid or solution form) , SMA in a second container (e.g. in solid or solution form) , and PAE/TPT in a third container (e.g. in solid or solution form) , and mixed and reconstituted before use.
Alternatively, any of the following forms can also be adopted: DMAA and SMA are mixed in a suitable ratio and placed in the first container, while PAE/TPT is present in the second container; DMAA and PAE/TPT are mixed in a suitable ratio and placed in the first container, and SMA in the second container; SMA and PAE/TPT are mixed in a suitable ratio and placed in the first container, and DMAA is present in the second container. These containers can be in a combined configuration, or completely separate.
In some further embodiments, the kit may comprise a container in which a mixture of DMAA, SMA and PAE/TPT is present, wherein the molar ratio of DMAA to SMA is in the range of 30: 1 to  3: 1, e.g., in the range of 20: 1 to 3: 1, in the range of 15: 1 to 3: 1, in the range of 10: 1 to 3: 1, in the range of 9: 1 to 3: 1, in the range of 8: 1 to 3: 1, in the range of 7: 1 to 3: 1, in the range of 6: 1 to 3: 1, in the range of 5: 1 to 3: 1, in the range of 4: 1 to 3: 1, more specifically, the molar ratio of DMAA: SMA may be about 4: 1 or 3: 1; and, wherein the molar ratio of SMA: PAE/TPT is in the range of 10: 0.005 to 10: 3, such as 10: 0.05 to 10: 3, 10: 0.08 to 10: 3, 10: 0.1 to 10: 3, 10: 0.2 to 10: 3, 10: 0.3 to 10: 3, 10: 0.4 to 10: 3, 10: 0.5 to 10: 3.10: 0.6 to 10: 3, 10: 0.7 to 10: 3, 10: 0.8 to 10: 3, 10: 0.9 to 10: 3, 10: 1 to 10: 3, 10: 1.1 to 10: 3, 10: 1.2 to 10: 3, 10: 1.3 to 10: 3, 10: 1.4 to 10: 3, 10: 1.5 to 10: 3, 10: 2 to 10: 3, more specifically the molar ratio of SMA: PAE/TPT may be about 10: 0.05, 10: 0.08, 10: 0.1, 10: 0.5, 10: 1, 10: 1.1, 10: 1.2, 10: 1.3, 10: 1.4, 10: 1.5, 10: 1.6, 10: 1.7, or 10: 1.8.
Further, in certain embodiments, the precursors for forming the hydrogel are DMAA, SMA and PAE/TPT, and the molar ratio of DMAA: SMA: PAE/TPT is in the range of (40±20) : (10±5) : (0.005-2) , Such as about (40±15) : (10±3.5) : (0.4±0.2) , about (40±10) : (10±2.5) : (0.4±0.2) , about (40±5) : (10±2) : (0.4±0.2) , about (40±5) : (10±1.2) : (0.4±0.2) , about (40±5) : (10±1) : (0.4±0.1) . In some other embodiments, the precursors for forming the hydrogel are DMAA, SMA, and PAE/TPT, wherein, on a mole basis, if DMAA is 30-50 parts, SMA is 5-15 parts and PAE/TPT is 0.005-1.5 parts. Specifically, the molar ratio of DMAA: SMA: PAE/TPT can be about 30: 10: 1, 40: 10: 0.08, 40: 10: 0.4, 40: 10: 1, 40: 10: 1.2, or 40: 10: 1.5.
The kit may also comprise a container with an anchoring agent, which may be NAS or NSA. The kit may also comprise a container with a polymerization activator or accelerator to add to hydrogel precursor solution prior to polymerization. The polymerization activator or accelerator may be selected from, for example, VA-044, TEMED, potassium persulfate and APS.
The kit may also comprise one or more of the following: a container comprising a disulfide bond reducing agent (e.g., TCEP) ; a container comprising a fixative (e.g., paraformaldehyde) ; a container comprising a protein denaturation buffer (e.g., comprising SDS) ; a container containing a protein staining solution (e.g., Coomassie brilliant blue staining solution) ; and a container containing an enzyme for digesting proteins in the sample. Optionally, the kit also contains several commonly used buffers to facilitate practicing the methods of the present invention.
A container is understood to mean any structure that can accommodate or surround the components of the hydrogel formulation, anchoring agent, etc. ; exemplary containers include bottles (e.g., plastic or glass bottles) , syringes, vials, sachets, capsules, ampoules, cartridges, etc. Containers can be shielded from visible, ultraviolet, or infrared radiation by using other components (for example, an aluminum foil bag around the vial) or by choosing the material properties of the container itself (for example, an amber glass vial or an opaque plastic bottle) .
The kit may also include a mixing device for mixing the precursors together to produce the hydrogel formulation of the present invention. The kit may also include gel manipulation devices, such as soft brushes, forceps, and/or delivery devices (which may or may not include mixing elements) , etc., for infusing the hydrogel solution onto the sample or transferring the gel.
In some embodiments, the kit includes one or more of the following components: a gelation chamber configured to accommodate the sample prior to gelation, e.g., a dish with a chamber or groove; a gel manipulation tool, e.g., tweezers , soft brush; a puncher; a cooling device, such as ice pack; multiple Eppendorf tubes, spin columns, etc.
The kit may also include other components, such as desiccants or other means of maintaining control of the water content in the kit, indicators to indicate the temperature of the kit, etc., which may be required to preserve the kit in good status during shipping and storage.
In some embodiments, the kit includes a gelation chamber for forming the sample-hydrogel composite as described above. The chamber is configured to have cavities, holes or grooves for accommadating the sample prior to gelation, e.g. in the form of a dish, e.g. a MatTek dish. The gelation chamber may also include a lid (e.g., a coverslip) that covers the cavity, well, or groove.
In some embodiments, the kit further includes an expansion chamber configured to expand the sample and accommodate the sample after gelation. The kit may include multiple vessels packaged together for ease of use.
In addition to the components described above, the kit will generally include instructions for using the components of the kit to carry out the methods of the present invention. Instructions for carrying out the methods of the present invention are typically recorded on a suitable recording medium. For example, the instructions may be printed on a material such as paper or plastic. Thus, the instructions may be present in the kit as a package insert, in the label of the container of the kit or its components. In some other embodiments, the instructions are stored as electronically stored data files on a suitable computer-readable storage medium, e.g., the actual instructions are not present in the kit, but are retrieved from a remote source (e.g., a compact disc) . An example of the embodiment is a kit that includes a website where instructions can be viewed and/or downloaded. As with the instructions, the means for obtaining the instructions are documented on suitable materials.
The ProteomEx method as disclosed herein provide several advantages including for instance:
1. Provide improvements to sampling and analysis methods for spatial proteomic identification, enabling more precise information about proteins at specific locations in a sample;
2. In the process of protein processing, more effective protein fragments and lower missing cleavage percentages can be obtained compared to standard methods in the field; and
3. Can be carried out conveniently by kits in a low-cost manner, and for ordinary technicians, the operation difficulty is significantly reduced compared with other spatial proteomics identification methods.
The following examples are provided to better illustrate the claimed invention and are not to be construed as limiting the scope of the invention. All of the following specific compositions, materials and methods fall in whole within the scope of the present invention. These specific compositions, materials and methods are not intended to limit the invention, but merely to illustrate specific embodiments that fall within the scope of the invention.
Examples
Preparation of reagents and materials
PFA (4%paraformaldehyde fixative solution)
2.5 ml of 16%paraformaldehyde fixative, 1 ml of 10 X PBS (phosphate buffered saline) , plus 6.5 ml of distilled water, stored at room temperature.
40X borate buffer (50 ml)
3.1 g of boric acid (final concentration 1 mol) , 1 g of sodium hydroxide (final concentration 0.5 mol) were weighed, adding distilled water to 50 ml, and store at room temperature.
TCEP-HCl, tris (2-carboxyethyl) phosphine hydrochloride solution (0.5 mol)
5.73 g of tris (2-carboxyethyl) phosphine hydrochloride were weighed, adding 25 ml of distilled water to dissolve, adjusting to pH 7 with 5 moles of sodium hydroxide, then adding distilled water to 40 ml, and stored at -20℃.
Disulfide bond reduction solution
1 mL of 40X boric acid buffer, 4 mL of 0.5 mol tris (2-carboxyethyl) phosphine hydrochloride solution, adding 35 mL of distilled water, and stored at 4℃.
MES buffer
100 mM MES (2- (N-morpholino) ethanesulfonic acid) , pH 6, dissolved in distilled water and stored at 4℃.
Protein anchoring solution
0.1 mg/ml acrylate-N-succinimidyl ester (NSA) dissolved in 100 mM MES buffer and stored at 4℃.
MOPS buffer
100 mM 3-morpholinepropanesulfonic acid buffer, pH 7, stored at 4℃.
Pentaerythritol allyl ether (PAE) solution
0.115 g/ml, 0.4 μM, dissolved in tetrahydrofuran, sealed and stored at 4℃.
Hydrogel precursor solution
N, N-Dimethacrylamide (DMAA) , Sodium Methacrylate (SMA) , and Pentaerythritol Allyl Ether (PAE) are formulated in a molar ratio of 40: 10: 0.4, for example, weighing 3.137 g of DMAA, 0.8624 g of SMA, adding 77 μl of PAE solution, 350 μl of 10%hydrochloric acid, and 4.6615 μl of distilled water to prepare a monomer solution, and stored at 4℃.
Activated hydrogel precursor solution
Pipetting 900 μl of the precursor solution, adding 30 μl of freshly prepared 10%ammonium persulfate (APS) , 20 μl of 10% (w/w) tetramethylethylenediamine (TEMED) , and then adding 50 μl distilled water to formulate the activated monomer solution, ready-to-use.
Protein denaturation buffer
28.7 g of sodium dodecyl sulfonate (final concentration 0.2 mol) , 1.545 g boric acid (final concentration 50 mmol) were weighed, adding distilled water to 500 ml, well mixed, and stored at room temperature.
Rapid Coomassie Brilliant Blue staining solution
8 ml of commercially available Coomassie brilliant blue concentrate was added with distilled water to 1 liter and stored at room temperature.
Example 1: Testing of hydrogel precursor solution
The expansion factor and mechanical strength of hydrogels formed from hydrogel precursor solutions containing different ratios of SMA: DMAA: PAE were tested. The Polymerization initiator used were VA-044 or KPS (potassium persulfate) . The inventors found that the combination of SMA+DMAA+PAE resulted in gels with the desired expansion factor and mechanical strength across a wide range of ratios (Table 1) , indicating that combinations of different ratios of SMA+DMAA+PAE are suitable for successful formulation into a hydrogel. The combination of SMA+DMAA+PAE reached similar effect. The expansion factor is a linear ratio calculated by comparing the sizes (e.g. length and width) of the gelated sample before and after expansion. After adding pure water to swell to the maximum extent, if the gel does not break, it is judged that the mechanical strength of the gel is good. The expansion factor and mechanical strength both as desired was evaluated as achieving a good effect
Table 1
Figure PCTCN2022115456-appb-000001
Figure PCTCN2022115456-appb-000002
Figure PCTCN2022115456-appb-000003
* Used in Examples 3 and 4 for comparison of ProteomEx with other methods
Example 2: Sampling of the expanded sample-hydrogel composite of the brain slice and MS analysis
1. Disulfide bond reduction
200 μl of PBS (phosphate buffer solution) was added to the chamber of a 35 mm laser confocal dish, and small soft brush was used to transfer a 30 μm brain slice fixed with 4%PFA (paraformaldehyde) to the chamber. Then the PBS buffer in the chamber was aspirated, and 200 μl of disulfide bond reduction solution (containing 50 mmol of TCEP-HCl) was added. The dish was covered and incubated at room temperature for 2 hours.
2. Protein anchoring
The disulfide bond reduction solution was removed, then the chamber was rinsed three times with 100 mM MES buffer, and incubated with 200 μl of protein anchoring solution for more than 6 hours at room temperature. The chamber was rinsed three times with 200 μl of 100 mM MOPS buffer.
3. Gelation (PAE-0.5)
The chamber was incubated in 200 μl of activated monomer ATMS solution (activated precursor solution containing APS and TEMED) at 4 ℃ for more than 6 hours; the solution was removed and a soft brush was used to plate the tissue section onto the bottom of the confocal dish, dried to make the tissue slice closely attached to the bottom of the chamber; then the tissue slice in the chamber was covered with a glass sheet several mms thick, leaving a small gap to add the activated precursor solution again, and incubated at room temperature for about 30 minutes. The covered laser confocal small dish was put into a vacuum drying oven sprinkled with distilled water and preheated to 37℃. A vacuum pump was used to evacuate the vacuum drying oven to a semi-vacuum, and then passing nitrogen to normal atmospheric pressure level, repeated for 4 times. The radical polymerization reaction was conducted for 1.5 hours to fully polymerize the activated precursor solution so as to form a hydrogel.
4. Protein Denaturation (SDS) and De-anchoring
The formed gel was transferred to a 6-well plate, 8 ml of protein denaturation buffer was added, and incubated at 95℃ for 3 hours. The gel was then transferred to a 6 cm dish, 10 mL of 50 mM tris buffer pH 8.8 was added, and was eluted at room temperature three times with 0.5 h each time.
5. Methanol elution
50%methanol aqueous solution was added, and eluted three times at room temperature for about 20 minutes each time.
6. Coomassie Brilliant Blue Staining and Expansion
10 ml of fast Coomassie brilliant blue staining solution was added and incubated at 95℃ for more than 1 hour until the gel color turns dark blue. Then the gel was transferred to a 10 cm dish, distilled water was added and incubated at 95℃ for 1 hour. The gel was eluted three times until the area other than the tissue became transparent. A tissue sample with a linear expansion of about 5 times was obtained (Fig. 3) .
7. Micro-tissue sampling
A 3 mm hole puncher was used and tiny areas of target tissue were selected for punching. The positions and numberings in the order shown in Figure 2 were recorded. Each piece of tissue was placed separately in a 1.5 ml EP tube and corresponding brain region in the brain map was recorded. After sampling, the samples were placed at -4℃ for subsequent manipulations.
8. Methanol cleaning
100 uL of 50%methanol was added to each EP tube and incubated at 25 ℃ for 15 min with spinning at 600 rpm, then the methanol solution was discarded, washed with ddH2O, and the above steps were repeated three times.
9. Digestion
Trypsin was diluted to a final concentration of 12.5 ng/μl by adding 25 mM ammonium bicarbonate (ABB) . The trypsin solution was added to each tube and incubated at 37℃ for 4 hours, then the trypsin solution was added again to each tube and incubated at 37℃ for 12 hours.
10. Peptide extraction (at room temperature)
After centrifuged at 1000 g for 3 min, the solution was collected into a new Eppendorf tube (Solution A) . 150 ul of 25 mM ABB was added and centrifuged at 600 rpm for 10 min, then the solution was collected into the corresponding Eppendorf tube (Solution B) . 150 ul of 50%ACN/50%25 mM ABB was added and incubated at 600 rpm for 10 minutes, then the solution was collected in the same Eppendorf tube (Solution C) . 150 ul of 50%ACN/50%ddH 2O/2.5%FA was added and incubated at 600 rpm for 30 minutes, then the solution was collected in the above Eppendorf tube (solution D1) . 150 ul of 50%ACN/50%ddH2O/2.5%FA was added and incubated at 600 rpm for 10 minutes. The solution was collected accordingly (Solution D2) . 150 ul of 100%ACN was added  and incubated at 600 rpm for 10 minutes. The solution (Solution E) was collected similarly. This step was repeated until the gel turns white and sticky. All solutions (solutions A, B, C, D1, D2, and E) were combined and centrifuged at 15, 000 g for 10 min, and the supernatant was collected in a new Eppendorf tube.
Peptide drying: The vacuum pump was preheated for 20 minutes, and final drying was performed at 40℃, 6-7 mbar for 4 hours, then stored at -20℃.
Peptide solubilization: Peptides were solubilized with 30 ul of 2%ACN/0.1%TFA.
11. Desalination
Micro Spin columns were placed in a new long EP tube, and washed with 20 μl MeOH by centrifugation, then placed in a new long EP tube, centrifuged and washd with 20 μl 80%ACN 0.1%TFA, equilibrated with 20 μl 2%ACN, loaded with 30 μl of peptide solvent, and centrifuged. Washed with 20 μl 2%ACN again and centrifuged.
Elution: Cup (with final label) was replaced and eluted twice with 30 μl 40%ACN 0.1%TFA, centrifuged at 1200 rpm for 5 min. Samples were dried in a SpeedVac. Centrifuged at 6-7 mbar for 4 hours at 45℃.
12. Re-dissolution
Peptides were re-dissolved in 10 ul of MS buffer (2%ACN, 0.1%FA) followed by centrifugation. The supernatant was then transferred to vials for mass spectrometry detection.
13. Data collection
8 μl (8/10) peptide solution was injected into HPLC-MS/MS (timsTOF) over 90 minutes.
HPLC setup
Buffer A: 100%H 2O, 0.1%FA
Buffer B: 100%ACN, 0.1%FA
Trap column: Thermo, 3um
MS column: BPRC, 1.9 um, 15 cm
Flow rate: 0.3 μL/min
Gradient: 5%-27%Buffer B (0-80 min) , 27%-40%Buffer B (80-90 min) , 40%-80%Buffer B (90-92 min) , 80%-80%buffer B (92-95 min) .
14. Data query
Data identification was performed using MS Fragger (V13.0) .
15. In-solution enzymatic hydrolysis (control)
Mouse brain slice samples were collected, added with Tris HCl pH 10, and conducted hydrolysis reaction at 95℃ for 30 minutes. Then a lysis solution was added, and lysis was conducted under ultrasonic conditions. A reducing reagent was added to the cleaved protein first,  and then incubated at room temperature for 30 minutes in dark, after which an alkylating reagent was added and reacted at 25℃ for 30 minutes. Next, trypsin was added to carry out the digestion reaction for a total of 16 hours. At the end of the digestion, 0.1%trifluoroacetic acid was added to terminate the digestion and desalted. The obtained peptide samples were subjected to HPLC-MS/MS for data-dependent acquisition (DDA) . Finally, the proteomic data was de-spectrified by MSFragger software and the number of identified proteins was analyzed.
16. Pressure Cycling Enzymatic Hydrolysis (Control)
Mouse brain slice samples were collected and after adding lysis solution, the pressure cycle technology (PCT) was used for lysis. 90 cycles of hydrolysis under the cycle program of 50 s, 45,000 psi and 10 s atmospheric pressure were performed. The hydrolyzed proteins were added with reducing and alkylating reagents for reaction. Next, lysC and trypsin were added for PCT-assisted digestion, and the conditions were set to 120 cycles, each cycle consisting of 50 s, 20, 000 psi and 10 s normal pressure. At the end of the digestion, 0.1%trifluoroacetic acid was added to terminate the digestion and desalted. The obtained peptide samples were subjected to HPLC-MS/MS for data-dependent acquisition (DDA) . Finally, the proteomic data were de-spectrified by MSFragger software with default parameters and the number of identified proteins was analyzed.
17. Results
Table 2 shows the yield of peptides per mg of brain slice samples after expansion gel digestion, in-solution digestion, or pressure cycling digestion, respectively. The yield of peptides is the amount of peptides extracted per mg of tissue. From the results, it can be found that the peptide yield of 48.36 μg/mg obtained by expansion gel digestion is much higher than that of traditional in-solution digestion (26.01 μg/mg) , and is also higher than the efficiency of pressure cycling digestion (32.31 μg/mg) . This method demonstrates the high efficiency of expansion gel enzymatic digestion and can effectively extract peptides for subsequent proteomic analysis. Figure 4 shows the number of polypeptides obtained by sampling the expanded hydrogel with a 3mm puncher and the number of identified protein species.
Table 2
Figure PCTCN2022115456-appb-000004
Figure 5 shows the corresponding number of identified protein species (Figure 5A) and the proportion of missing cleavage after three identical brain slice samples were subjected to expansion gel digestion, in-solution digestion or pressure cycling digestion (ie, PCT-assisted digestion) (Fig. 5B) . The missing cleavage percentage is the percentage of the number of missing cleaved peptides to the total number of peptides identified by the software. The results of the comparison of the three methods show that the expansion gel digestion can obtain comparable identification results in the number of protein species to the other two methods, and has the lowest missing cleavage percentage. The standard deviation of the results obtained by expansion gel digestion was smaller and the stability was better (Fig. 5A) . Meanwhile, the proportion of missing cleavage was significantly reduced, and the efficiency of trypsin digestion was increased (Fig. 5B) .
Example 3. Comparison of ProteomEx with other common methods in the field in terms of peptide yield, trace sample size, miss cleavage rate
Since ProteomEx involves chemical treatment and expansion of tissue, it is important to quantify the efficiency of peptide extraction and validate the qualitative and quantitative reproducibility and sensitivity achievable with this method. First, we decided to measure peptide yield and missed cleavage rate as the major parameters of sample preparation quality control. For benchmarking, we used the well-established in-solution digestion and the pressure cycling technology (PCT) -assisted tissue digestion methods, which are widely used for tissue treatment in MS-based proteomics analysis. We also used a proExM-MS method for benchmarking.
Tissue expansion for proExM-MS was performed according to the following protocol: First, PFA-fixed mouse brain tissue was treated with succinimidyl ester of 6- ( (acryloyl) amino) hexanoic acid (AcX) , 0.1 mg/mL in PBS overnight in a humid chamber at 22℃. Freshly prepared monomer solution (8.6% (w/v) sodium acrylate, 30% (v/v) acrylamide/bisacrylamide (30%solution; 37.5: 1) , 2 M NaCl, 0.01% (w/v) 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-1-oxyl (4-hydroxy TEMPO) inhibitor dissolved in 1x PBS and supplemented with 0.2% (w/v) TEMED, and 0.2% (w/v) APS) was deposited on the tissue slice and evenly spread inside a gelation chamber then covered with glass slide and incubated for 2h at 4℃. Polymerization was carried out in vacuum oven (DZF-6000, Shanghai Sunrise Instrument) at 37℃ for 2 h. Homogenization was performed with 5%SDS in water incubated at 58℃ in a humidity chamber overnight. For expansion, gels were rinsed in water and placed in excess volumes of doubly deionized water for ～1 h to expand while exchanging water every 15 min. No Coomassie staining was used for proExM-MS samples.
Processing of the adjacent mouse brain tissue sections using the selected methods revealed that peptides extraction yield for ProteomEx was about 1.4-1.7-fold higher than that for in-solution  digestion, PCT, and proExM-MS, specifically 72.54±6.17 μg peptides/mg tissue (mean ± standard deviation (SD) , throughout unless otherwise indicated for ProteomEx vs. 44.59±18.54 μg, 43.58±11.59 μg, and 50.78±9.33 μg for in-solution, PCT, and proExM-MS, respectively; Figure 6A) . Furthermore, ProteomEx was characterized by a lower number of missed cleavages (20.4±1.0%) compared to 27.96±1.4%and 24.07±1.2%for in-solution and PCT protocols, respectively, although similar to that for proExM-MS (21.1±5.4%; Figure 6B) . The higher efficiency of peptide digestion and extraction achieved with ProteomEx and proExM-MS can be probably explained by molecular decrowding in the expanded state providing better access for enzyme molecules to the proteolytic sites. These results indicated that the tissue expansion protocol used in ProteomEx provided higher efficiency of peptide extraction compared to in-solution and PCT-assisted sample preparation methods for tissue samples as well as the conceptually similar proExM-MS.
Next, the extracted peptides were analyzed using a timsTOF Pro mass spectrometer in data-dependent acquisition (DDA) mode. By processing ～200 ng of peptides from each sample, we identified 37, 173, 34, 304, 20, 413, and 30, 630 peptides corresponding to 4199, 4278, 3181, and 3818 individual proteins on average per sample prepared using in-solution, PCT, proExM-MS, and ProteomEx methods, respectively (Figure 6C, D) . Variability of peptide and protein numbers were lower for ProteomEx, proExM-MS, and PCT than that for the in-solution method, suggesting a higher degree of reproducibility of these methods. The number of proteins identified with ProteomEx was lower by about 400 proteins than that identified with in-solution and PCT methods, though higher by 637 proteins than that for proExM-MS.
The diversity of all identified proteins with the four methods had an overlap of 56.6%, and each method uniquely identified less than 7.0%of the total number of proteins (Figure 6E) . The overlap for the identified proteins was reasonable (>50～60%is the typical overlap for DDA mode for data analysis) and high enough to indicate that ProteomEx performance was comparable to the methods used for sample processing in MS-based proteomics. Furthermore, all four methods exhibited similar distribution of identified proteins by biomarkers, subcellular localization, and biological function (Figure 6F) . Overall, the above results demonstrated that ProteomEx could acquire a high-quality proteome and was comparable with the other available methods in terms of the number and type of protein identification, which met the proteomic analysis needs.
Staining of the expanded tissue with the colorimetric dye facilitated visualization of fine morphological features with the naked eye and precise targeting of a region of interest by manual microdissection (down to ～100 μm of real size) . To validate this capability for MS analysis, we used a biopsy punch, which provided highly reproducible microsampling, to excise 3 mm-diameter  tissue-hydrogel composite pieces (corresponding to ～500 μm in diameter or 5.9 nL tissue volume before expansion) and adjacent blank hydrogel pieces (used as a control of possible peptide diffusion outside of tissue) from the same expanded mouse brain tissue slice and analyzed as described above using pure MS buffer as a negative control. We identified 24, 437/3541 peptides/proteins on average per punched sample, which was much higher than that for the blank hydrogel (416/132 peptides/proteins) and MS buffer (260/116 peptides/proteins corresponding to carry-over level; Figure 6C, D) , and similar protein distribution by subcellular localization and biological function as observed for the bulk sample analysis (Figure 6F) . The ProtemoEx method showed comparable reproducibility of protein quantification in macro-and microsample preparations of brain tissue compared to other tested methods (Figure 7) . Furthermore, ProteomEx did not introduce chemical modifications to the extracted peptides and exhibited similar to other methods in the qualitative and quantitative of peptide post-translational modifications (Figure 8) . Thus, peptides can be efficiently extracted from the small pieces of the expanded tissue with neglectable diffusion into the blank hydrogel around the tissue. Additionally, compared with whole-brain slices processed by ProteomEx, 3 mm gels identified a slightly lower number of peptides while a comparable number of proteins.
Next, we explored the volume-dependent limit of tissue microsampling using ProteomEx approach. Processing the microdissected mouse brain coronal sections with actual volume of about 0.6, 2.4, 5.4, 9.6, and 15.0 nL (corresponding to lateral resolution of about 160, 320, 480, 640, 800 μm) , we identified 2987, 15, 705, 23, 898, 35, 160, and 37, 071 peptides corresponding to 928, 3044, 4203, 5058, and 5105 unique proteins, respectively, on average per size group (Figure 6G, H) . As expected, the numbers of identified peptides and proteins increased with tissue volume reaching a plateau at around 5.0 nL tissue size (or 480 μm in diameter) . PCT-assisted sample preparation, as a representative method for processing small samples, enables effective analysis of tissues volume in the range of 0.2-1 μL, which is about three orders of magnitude higher than ProteomEx. Albeit it was more peptides and proteins identified by PCT due to a larger sample injection amount, ProteomEx showed a higher degree of reproducibility in the processing of small sample volumes compared to PCT (Figure 6G, H) . ProteomEx provides a new strategy for sub-nanoliter volume sample preparation for proteomic analysis.
Example 4 ProteomEx can be applied to multiple biological tissues with expansion artifacts.
To assess the applicability of ProteomEx to various mammalian tissues, we performed ProteomEx on three different mouse tissue types including brain, liver, and breast cancer (Figure 9A) . Since ProteomEx utilized novel hydrogel composition and optimized homogenization treatment, we first quantified the isotropy of hydrogel-based tissue expansion using a non-rigid  registration as done previously for the original protein-retention ExM method (Tillberg et al., Nature Biotechnology, 2016) . The isotropic expansion is essential for precise mapping of spatial proteome distribution onto pre-expanded tissue morphology. We calculated the root-mean-square (RMS) length measurement error of feature measurements after tissue expansion over length scales up to 1500 μm and found that RMS errors were ～8%, ～10%, and ～8%of the measurement distance for brain, liver, and breast cancer tissue samples, respectively (Figure 9B) . Next, we processed ～5 nL volume of each tissue type using DDA-MS and identified 24, 436/3540, 14, 298/2606, 9623/2356 peptides/proteins for brain, liver, and breast cancer samples, respectively (Figure 9C, D) . To explore the possibility to correlate proteomic profile with cellular and subcellular features visualized via immunohistochemistry and small dye staining, we stained the AD mouse brain slice with DAPI and Aβ antibodies, imaged and processed it using ProteomEx. For the 2.52 nL volume of the immunostained tissue, we identified 7000 peptides corresponding to 2000 proteins for three replicates demonstrating the compatibility of ProteomEx with immunohistochemistry. ProteomEx can be readily applied to different mammalian tissue types and is compatible with antibody-stained samples.
To explore the limits of lateral spatial resolution and tissue volume for ProteomEx, we expanded the brain tissue section by 8-fold in linear dimension (512-fold in volume) and punched out 1-mm radius tissue-hydrogel composite corresponding to the pre-expansion radius of 125 μm for proteomic analysis. The pre-expansion volume of punched tissues was 0.37 nL, equivalent to approximately 160 cells (calculated using BNID 100434) . On average, we identified and quantified more than 3000 peptides and 1000 proteins per sample analyzed in PulseDIA mode (Figure 9E) .

Claims (37)

  1. A method for processing a biological sample for protein identification, comprising:
    treating the biological sample with an anchoring agent, adding a hydrogel precursor solution and polymerize, thus forming a sample-hydrogel composite;
    performing protein denaturation and de-anchoring on the sample-hydrogel composite;
    performing protein staining on the sample-hydrogel composite, and let the composite expand; and
    sampling from the expanded sample-hydrogel composite for protein identification.
  2. The method of claim 1, wherein the biological sample is selected from a specimen, a tissue slice, a biopsy sample, an organ or part thereof, and an entire organism.
  3. The method of claim 1 or 2, wherein adding the hydrogel precursor solution is infusing the biological sample with the hydrogel precursor solution or embedding the biological sample in the hydrogel precursor solution.
  4. The method of any of claims 1-3, wherein the anchoring agent is added into the hydrogel precursor solution, thereby anchoring and polymerizationare performed simultaneously.
  5. The method of any of claims 1-4, wherein the anchoring agent comprises a biomolecule reactive chemical group and a hydrogel reactive chemical group.
  6. The method of claim 5, wherein the anchor is selected from acrylate-N-succinimidyl ester (NSA) , N- (allyloxycarbonyloxy) -succinimide (NAS) and combinations thereof.
  7. The method of any of the preceding claims, wherein the hydrogel precursor solution comprises one or more precursor (s) of hydrogel polymer.
  8. The method of claim 7, wherein precursor is selected from sodium acrylate (SA) , sodium methacrylate (SMA) , itaconic acid (IA) , trans-aconitic acid (TAA) , ethyl-2- (hydroxymethyl) -acrylate (EHA) , N, N-methylenebisacrylamide (MBAA) , N, N-dimethylacrylamide (DMAA) , pentaerythritol tetraacrylate (PT) , propoxylated trimethylol Propane triacrylate (TPT) ,  pentaerythritol triacrylate (PA) , dipentaerythritol pentaacrylate or dipentaerythritol hexaacrylate (DPHA) , trimethylolpropane triacrylate (TTA) , bis (trimethylol) Propane) -tetraacrylate (DiTA) , trimethylolpropane trimethacrylate (TTMA) , glycerol propoxylated (1PO/OH) triacrylate (GPT) , ethoxylated trimethylolpropane Triacrylate (TET) , pentaerythritol allyl ether (PAE) , sodium 4-hydroxy-2-methylenebutyrate (SHMB) , N, N-dimethylaminopropylacrylamide (DMPAA) and acrylamide (AA) .
  9. The method of claim 8, wherein the precursors comprised in the hydrogel precursor solution are SMA, DMAA and PAE, optionally the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1, the molar ratio of SMA: PAE is within the range of 10: 0.005 to 10: 3, preferably the molar ratio of DMAA: SMA: PAE is (30-50) : (5-15) : (0.005-2) .
  10. The method of claim 8, wherein the precursors comprised in the hydrogel precursor solution are SMA, DMAA and TPT, optionally the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1, the molar ratio of SMA: TPT is within the range of 10: 0.005 to 10: 3, preferably the molar ratio of DMAA: SMA: TPT is (30-50) : (5-15) : (0.005-2) .
  11. The method of any of the preceding claims, wherein prior to adding the precursor hydrogel solution, a polymerization activation agent and/or polymerization promoting agent, such as VA-044, V50, ammonium persulfate, potassium persulfate and TEMED, is added into the precursor hydrogel solution.
  12. The method of any of the preceding claims, wherein method further comprises treating the biological sample with a disulfide bond reductant, and the disulfide bond reductant is selected from TCEP, DTT, and BME.
  13. The method of any of the preceding claims, wherein the protein degeneration is performed by treating with a detergent (such as SDS or sodium dodecyl benzene sulfonate) .
  14. The method of any of the preceding claims, wherein the de-anchoring by performed by increasing pH value to alkaline (for example, by contacting Tris buffer) to break the amide bond.
  15. The method of any of the preceding claims, wherein after protein de-anchoring and prior to protein staining, the method further comprises washing the sample-hydrogel composite with an organic solvent such as methanol.
  16. The method of any of the preceding claims, wherein the protein staining is selected from Coomassie blue staining, Coomassie brilliant blue staining, immunostaining, silver staining, and fluorescence staining.
  17. The method of any of the preceding claims, wherein the expansion of the sample-hydrogel composite is performed by incubating the composite with water or an aqueous buffer.
  18. The method of any of the preceding claims, wherein the linear extension factor of the expanded sample-hydrogel composite is 5-7.
  19. The method of any of the preceding claims, wherein the sampling is local sampling, such as micro-dissection or local sampling via a punch.
  20. The method of claim 19, wherein the diameter of the sample (s) obtained from local sampling is 0.1-3 mm.
  21. The method of any of the preceding claims, wherein protein identification is performed by mass spectrometry analysis, such as HPLC-MS.
  22. The method of claim 21, wherein the mass spectrometry analysis comprises performing enzymatic digestion, peptide extraction (optionally demineralization) and mass spectrometry detection on the sample-hydrogel composite.
  23. Ahydrogel formulation comprising a combination of DMAA, SMA and PAE, or a combination of DMAA, SMA and TPT.
  24. The hydrogel formulation of claim 23, wherein the formulation comprises DMAA, SMA and PAE, and the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1, the molar ratio of SMA: PAE is within the range of 10: 0.005 to 10: 3, preferably the molar ratio of DMAA: SMA: PAE is (20-60) : (5-15) : (0.005-2) ; or
    the formulation comprises DMAA, SMA and TPT, and the molar ratio of DMAA: SMA is within the range of 30: 1 to 3: 1, the molar ratio of SMA: TPT is within the range of 10: 0.005 to 10: 3, preferably the molar ratio of DMAA: SMA: TPT is (20-60) : (5-15) : (0.005-2) .
  25. The hydrogel formulation of claim 23 or 24, wherein the hydrogel formulation is prepared as a stock solution of DMAA, SMA, and PAE diluted in water, or DMAA, SMA, and TPT diluted in water.
  26. The hydrogel formulation of claim 23 or 24, wherein the hydrogel formulation is prepared as a mixture of DMAA, SMA, and PAE without dilution, or a mixture of DMAA, SMA, and TPT without dilution.
  27. Use of the hydrogel formulation of any of claims 23-26 for expansion of a biological sample.
  28. A kit, comprising DMAA, SMA and PAE in one or more containers, or DMAA, SMA and TPT in one or more containers.
  29. The kit of claim 28, wherein DMAA, SMA, and PAE each are in separated containers, optionally, DMAA and SMA are in solid form while PAE is in liquid form solubilized in an organic solvent.
  30. The kit of claim 28, wherein at least two of DMAA, SMA, and PAE are mixed in one container.
  31. The kit of claim 30, wherein DMAA, SMA, and PAE are mixed together in a molar ratio in the range of (20-60) : (5-15) : (0.005-2) , such as (40 ± 10) : (5-15) : (0.005-2) , (40 ± 15) : (10 ± 3.5) : (0.4 ± 0.2) , (40 ± 10) : (10 ± 2.5) : (0.4 ± 0.2) , (40 ± 5) : (10 ± 2) : (0.4 ± 0.2) , (40 ± 5) : (10 ± 1.2) : (0.4 ± 0.2) , (40 ± 5) : (10 ± 1) : (0.005 ± 0.1) , optionally DMAA, SMA, and PAE are dissolved in water and formulated as a solution.
  32. The kit of any of claims 28-31, wherein the kit is suitable for storage at 0℃-4℃ or at room temperature.
  33. The kit of any of claims 28-32, wherein the kit further comprises one or more containers comprising an anchoring agent such as NAS and/or NSA.
  34. The kit of any of claims 28-33, wherein the kit further comprises one or more of the following:
    a container comprising a disulfide bond reductant (such as TCEP) ;
    a container comprising a polymerization activation agent or polymerization promoting agent, such as VA-044, V50, TEMED, ammonium persulfate and potassium persulfate;
    a container comprising a fixative (such as paraformaldehyde) ;
    a container comprising a protein denaturation buffer (such as a protein denaturation buffer comprising SDS or sodium dodecyl benzene sulfonate) ;
    a container comprising protein staining solution (such as Coomassie blue staining buffer) ; and
    a container comprising enzymes (such as trypsin) for digestion of proteins in the sample.
  35. The kit of any of claims 28-34, wherein the kit further comprises one or more of the following:
    a gelation room configured to accommodate the sample before gelation, such as a dish with cavity or grooves;
    a staining room configured to stain the proteins in the sample;
    a hydrogel operation tool, such as a tweezer, soft brush;
    a punch; and
    a cooling device, such as ice bags.
  36. The kit of any of claims 28-35, which is used for the expansion of a biological sample.
  37. A system for spatial proteomics analysis of a biological sample, comprising:
    an anchoring module for anchoring the proteins in the biological sample;
    a gelation module for hydrogel polymerization to form a sample-hydrogel composite;
    a protein degeneration module for denaturation of the proteins in the sample-hydrogel composite;
    a protein de-anchoring module for de-anchoring of the proteins in the sample-hydrogel composite;
    a staining module for protein staining and optionally local sampling of the sample-hydrogel composite; and
    optionally, mass spectrometry analysis module for pre-processing of the samples obtained by local sampling before mass spectrometry and which is connected with a mass spectrometry detection device.
PCT/CN2022/115456 2021-08-27 2022-08-29 Methods and formulations for expansion proteomic analysis of biological samples WO2023025318A1 (en)

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