Protocol to characterize basement membranes during kidney development using mass spectrometry-based label-free quantitative proteomics

Summary Basement membranes are specialized extracellular matrices formed by highly insoluble structural proteins and extracellular matrix (ECM)-bound components that provide structural and signaling support to tissues and are dynamic during development. Here, we present a mass spectrometry-based label-free quantitative proteomics protocol to investigate basement membranes and define their composition using samples from human kidney organoids and mouse fetal kidneys. This protocol facilitates the study of basement membrane and other ECM components during development to improve our understanding of matrix regulation and function. For complete details on the use and execution of this protocol, please refer to Morais et al.1


SUMMARY
Basement membranes are specialized extracellular matrices formed by highly insoluble structural proteins and extracellular matrix (ECM)-bound components that provide structural and signaling support to tissues and are dynamic during development.
Here, we present a mass spectrometry-based label-free quantitative proteomics protocol to investigate basement membranes and define their composition using samples from human kidney organoids and mouse fetal kidneys.This protocol facilitates the study of basement membrane and other ECM components during development to improve our understanding of matrix regulation and function.For complete details on the use and execution of this protocol, please refer to Morais et al. 1

BEFORE YOU BEGIN
3][4][5] The protocol below describes a streamlined framework to enrich fetal mouse kidneys and human iPSC-derived kidney organoids for basement membrane proteins, as well as other extracellular matrix proteins, through chemical fractionation and further deep proteome profiling with discovery label-free quantitative mass spectrometry.

Institutional permissions
All mouse experiments described here were performed under the approval of the Animal Ethics Committee of the Institute of Biomedical Sciences (University of Sa ˜o Paulo, protocol 019/2015).

Experimental design for sample collection for proteome profiling
Timing: 25 days for kidney organoid differentiation; 19 days for mouse fetal kidney acquisition For this protocol, we will describe the procedures done using human kidney organoids and fetal mouse kidneys (Figure 1A).a. Kidney organoids are generated from human pluripotent stem cells (PSCs). 6.Collect kidney organoids from inserts on days 14, 18, and 25, wash with PBS, snap-freeze in liquid nitrogen, and store at À80 C until further processing.2. Mouse mating schedule and sample acquisition.a.To obtain fetal mouse kidney samples, breed the mice by mating females and males for 12 h, and check on the next morning for detection of pregnancy, which is confirmed based on visualization of a vaginal plug.b.Keep pregnant females until the 19 th day of gestation and then anesthetize them with tribromoethanol (0.025 mL/g of body weight) to submit to caesarian surgery for collection of the fetuses.c.Dissect fetal mouse kidneys using clean forceps and surgical scissors, snap-freeze in liquid nitrogen, and store at À80 C until further processing.
CRITICAL: Discard used blades and/or needles used for dissecting the mouse samples in a biohazard sharps container.Ensure the use of adequate personal protective equipment, including a lab coat, gloves (e.g., latex, nitrile), face mask, and eye protection.

KEY RESOURCES TABLE
Figure 1.Image of key steps (A) Representative images of day 25 human kidney organoids, and E19 fetal mouse kidney with scale bars, both processed for extracellular matrix enrichment and further proteomic analysis with mass spectrometry.(B) Tissue homogenate (before and after incubation and centrifugation in lysis buffer) during fractionation for enrichment for matrix proteins.
REAGENT Let the beads settle down before using and pipette from the bottom.The bead solution can be kept at 20 C in a sealed glass bottle for several weeks.
Prepare a 1 M triethylammonium bicarbonate (TEAB) stock solution and adjust pH to 8.5 with phosphoric acid.
This solution can be frozen at À20 C and stored for several weeks.

STEP-BY-STEP METHOD DETAILS
Sample enrichment for extracellular matrix proteins Timing: 4-5 h (for step 1) Timing: 30-60 min (for step 2) (may vary according to the number of samples and sonication protocol) Timing: 1 h (for step 3) Timing: 30-60 min (for step 4) (may vary according to the number of samples) For label-free quantitative discovery proteomics, we employ a chemical fractionation strategy 12 to enrich fetal mouse kidneys and kidney organoids for the detection of extracellular matrix proteins.Although we describe here an application for mammalian kidney systems, this protocol has also been tested with different tissue types (e.g., skin, brain meninges, lungs, and heart) and organisms (e.g., Drosophila, C. elegans).For highly fibrous and/or keratinized tissues, additional steps may be necessary to ensure full solubilization of fractionation pellets.
CRITICAL: Ensure adequate use of personal protection equipment (lab coat, gloves, facial mask, safety glasses) throughout sample fractionation and following processing for mass spectrometry.
Note: Your samples were stored at À80 C, in sample tubes.a. Thaw kidney organoids and fetal mouse kidney samples and wash briefly with ice-cold PBS.
CRITICAL: Samples must be kept on ice all the time to minimize protein degradation.
Note: In this study, at least 3 kidney organoids per time point were collected and pooled whilst 6 mouse fetal kidney samples (2 kidneys per fetus, approximately 8 mg of tissue) were processed individually.b.Place the samples in clean Petri dishes and cut the samples into small pieces (roughly 1 mm 3 ) using a sterile blade.
Note: For adult kidney samples, remove the kidney capsule and pelvis prior to tissue homogenization as these compartments are fibrous and may interfere with the efficiency of the enrichment strategy.
CRITICAL: Discard used blades in a biohazard sharps container.
c. Add 0.5 mL-1 mL of ice-cold PBS to the tissue homogenate to aid in collecting and transferring it to 1.5 mL sample tubes.d.Incubate the samples for 5 min on a rotator to remove residual blood.e. Centrifuge at 10,000 3 g for 1 min at 4 C and discard the PBS.Repeat this step at least once until all PBS has been removed.f.Add freshly prepared Tris-lysis buffer to samples tubes.
i.For kidney organoids, add 50 mL of Tris buffer.
ii.For mouse fetal kidneys, add 100 mL-200 mL of Tris buffer.
Note: Use the smallest possible volume of Tris lysis buffer -high volumes might be difficult to handle when processing the samples for later trypsin digestion.Furthermore, very dilute samples will yield poor protein coverage in the proteomic analysis.We recommend a 5:1 (v/w) ratio for buffer and tissue, respectively.For approximately 3 mg-5 mg of tissue, 200 mL of lysis buffer might be enough for processing.
g. Mechanically disrupt samples through repetitive pipetting using 200 mL pipette tips.Remember to keep the bottom of the tube on ice.
Note: Consider using a syringe equipped with an 18-or 21-gauge needle to shear the partial homogenate by passing it through the needle, however, be careful with sample loss when using needles compared to pipette tips.
CRITICAL: Discard used needles in a biohazard sharps container.
h. Incubate the homogenate (Figure 1B) under gentle rotation (10 RPM) at 4 C to allow cell lysis and extraction of soluble proteins.
Note: Incubation may vary from 30 min for cell-based samples to 1 h for tissue samples.
i. Centrifuge the sample tubes at 14,000 3 g for 10 min at 4 C. j.Collect the supernatant and transfer it to new sample tubes and label it as Fraction 1 -This is your first sample fraction that is enriched for soluble cellular proteins.
CRITICAL: Be careful not to disturb the pellet while collecting the supernatant.
Note: Tubes containing Fraction 1 should be kept on ice while the fractionation is carried out or stored at À80 C until further processing.
k. Resuspend the pellet in a freshly prepared alkaline detergent buffer by repetitive pipetting.
Note: As for the Tris-lysis buffer, we recommend using the smallest possible alkaline buffer.
CRITICAL: If the pellet is not fully resuspended, use a syringe equipped with an 18-gauge needle to shear by passing the partial pellet through the needle.
l. Incubate under gentle rotation at 4 C for 30 min to 1 h to allow solubilization and disruption of cell-matrix interactions.
CRITICAL: Incubate the samples in the alkaline buffer for no longer than 1 h as the NH 4 OH will damage proteins.
m. Centrifuge the sample tubes at 14,000 3 g for 10 min at 4 C. n.Collect the supernatant and transfer it to new sample tubes and label it as Fraction 2 -This second sample fraction is less concentrated than Fraction 1 and contains cell surface and transmembrane proteins.
CRITICAL: Be careful not to disturb the pellet while collecting the supernatant.
Note: Tubes containing Fraction 2 should be kept on ice while the fractionation is carried out or stored at À80 C until further processing.
Note: Fraction 1 and Fraction 2 may be combined in a 1:2 (v/v) proportion as a Cellular Fraction or processed separately.
o. Resuspend the remaining pellet in 50 mL of 13 TEAB/SDS lysis buffer by repetitive pipetting, and label it as Fraction 3 -this is the enriched ECM fraction.It will be a visible pellet in a range of around 50 mL.
Note: At this stage, the pellet consists of a thick, viscous, transparent mixture of highly insoluble proteins, which may be difficult to resuspend completely into solution through manual homogenization.Proceeding to sonication will aid with dissolving the pellet.
Note: Collect a desirable volume of Fraction 1 and Fraction 2, or Cellular Fraction, and transfer to new sample tubes, and then add the same volume of 23 TEAB/SDS lysis buffer.The final concentration of SDS will be 5%, which will ensure protein denaturation and inactivation of undesirable protease digestion.

Note:
The user can opt to add 5 mM of dithiothreitol (DTT) to the samples at this stage to improve disulfide reduction later after the sonication step.
Note: Other sonication approaches might also be considered and optimized to yield full disruption of the pellet without physically damaging proteins.
Note: *In our study, the laser-captured fetal mouse glomeruli were not processed for matrix enrichment but resuspended in 13 TEAB/SDS lysis buffer and sheared by sonication.
c. Place the sonicated ECM sample fractions on ice.
Note: You may store the sample fractions in 13 TEAB/SDS lysis buffer at À80 C at this stage or proceed to the protein reduction and alkylation.a.To measure protein concentration, use Millipore Direct Detect Assay-free Cards and place 2 mL of (reduced, alkylated) protein lysate in the center of corresponding membrane positions sized spots (and 2 mL of blank, i.e., 13 TEAB/SDS lysis buffer, in the membrane designated as the blank position).b.Insert the card into a Millipore Direct Detect Spectrometer and read the corresponding protein concentration for each sample.
Note: This system requires only 2 mL per analysis to provide accurate measurement of protein lysates.If your sample is highly concentrated, consider diluting it in 13 TEAB/SDS lysis buffer added with 5 mM DTT and 15 mM IAM.Alternatively, other protein quantification approaches such as colorimetric protein assays (Bradford, BCA) can be considered.

Sample processing for label-free mass spectrometry-based proteomics
Timing: 2 h (for step 5) Timing: 16 h (for step 6) Timing: 1 h (for step 7) (may vary according to the number of samples) Timing: 2-4 h (for step 8) This section will detail the procedures for protein clean-up using ProtiFi S-Trap Spin Columns and subsequent protein digestion to peptides with trypsin.At this stage, the sample fractions consist of reduced, alkylated protein lysates in 50 mM TEAB (pH 7.5) containing 5% SDS.If the samples have been stored at À20 C, allow enough time for the samples to thaw at room temperature.
5. Day 2: Sample preparation for trypsin digestion in S-Trap Spin Columns.
Note: Before starting, we recommend processing 25 mL or 50 mL of sample fractions to facilitate calculations for sample washing, cleaning, and digestion.

Note:
The ProtiFi S-Trap micro spin columns are equipped with derivatized silica that traps undigested proteins within its submicron pores, which allows washes to fully remove contaminants and detergents.The micro spin columns are suitable for samples with 1 mg-100 mg of proteins as per the manufacturer's recommendations.
Note: For the following steps, we will consider a sample volume of 50 mL, but adjustments can be made to other volumes accordingly.
a. 50 mg of protein is needed as starting material (the volume of sample fraction required can be determined based on previous protein concentration measurements).

Note:
If not 50 mL of protein lysates, make it up to this volume with 13 TEAB/SDS lysis buffer.
b. Add 5 mL of 12% (v/v) phosphoric acid (final concentration will be 1.2% phosphoric acid) and vortex mix briefly.
Note: This step acidifies the lysate to pH < 1.0, which is critical to denature the proteins and aid the interaction with the resin within the spin columns.
c. Add 330 mL of protein binding/washing buffer and vortex mix briefly.d.Place the spin columns on top of 2 mL collecting tubes, and add the acidified, methanolic protein lysates into the columns.e. Centrifuge at 4,000 3 g for 2 min.
CRITICAL: Because the spin columns fit approximately 200 mL, sequential loading, and centrifugation rounds will be necessary if your sample volume is > 200 mL until all the lysate has passed through the silica.
Note: Do not let the liquid that passes through the spin column come in contact with the protein-trapping silica within the column.f.Wash the trapped proteins with 150 mL of MTBE/MetOH by simply adding the solution and centrifuging at 4,000 3 g for 2 min and discarding the flow-through.
Note: This wash with MTBE/MetOH will remove lipids that are not removed using methanol only.
g. Wash with 150 mL of protein binding/washing buffer by simply adding the buffer followed by centrifugation at 4,000 3 g for 2 min, and discard the flow-through.Repeat this wash at least three times.
Note: Protein denaturation through incubation with protein solvent (SDS), acidification, and multiple exposure to high concentrations of methanol will eliminate any undesired protease activity, hence reducing proteolysis significantly, and maximizing the efficiency of trypsin digestion next.Note: This volume will be enough for 20 reactions (consider using no less than 1 mg of the protease of choice per 10 mg of protein/spin column).
c. Add 20 mL of trypsin into the spin columns using a gel loading tip.
CRITICAL: When adding the trypsin solution, do not touch the trapping silica to avoid contamination.Do not leave air bubbles between the trypsin solution and the trapping silica -the S-Trap binding silica is highly hydrophilic and will absorb the trypsin solution.
d. Cap the spin columns to limit the evaporative loss and place the columns/collecting tubes on a heating block and incubate at 37 C for 12 h.
Note: Alternatively, incubate for 1 h at 47 C. Do not shake or move the columns during incubation.Note that some dripping may occur during incubation, but this is not of concern.
After the incubation finishes, proteins will have been fully (or partially) digested to tryptic peptides.The next steps will recover the hydrophobic peptides from S-Trap trapping silica.From now on, we will keep and combine each elution after each centrifugation round to yield the peptide samples.a. Remove the spin column and collecting tubes very carefully from the heating block and allow a few minutes for them to cool down to room temperature.b.Add 65 mL of digestion buffer to the spin columns and centrifuge at 4,000 3 g for 2 min and keep the flow-through.c.Add 65 mL of 0.1% aqueous formic acid and centrifuge at 4,000 3 g for 2 min.Combine the flow-through with the first elution.d.Add 30 mL of 0.1% formic acid in 30% ACN and centrifuge at 4,000 3 g for 2 min.Combine the flow-through with the previous elution.

Note:
The final elution volume is approximately 180 mL, and the final concentration of acetonitrile will be around 5% (v/v).
e.The peptide samples are in an acidic mixture of organic solvents and salts.Proceed to desalt or store the elution at 4 C for 12 h.e. Wash the beads with 200 mL of 0.1% formic acid and centrifuge at 200 3 g for 1 min to remove the liquid.Repeat this wash once.f.Add the peptide samples to the wells containing the beads and incubate on a plate mixer for 5 min at 300 RPM without heating.g.Centrifuge at 200 3 g for 1 min.
Note: Each well fits approximately 200 mL of liquid, hence, repeat the last two steps if there is an additional sample left to process.h.Add 200 mL of 0.1% formic acid to the wells containing the beads and peptides and incubate on a plate mixer for 2 min at 300 RPM without heating.i. Centrifuge at 200 3 g for 1 min and discard the flow-through.j.Repeat the wash once.k.Replace the 96-well collecting plate with a new plate with unused, clean wells, to elute the peptides.l.Add 50 mL of 0.1% formic acid in 30% ACN to the wells containing the beads and peptides and incubate on a plate mixer for 2 min at 300 RPM without heating.m.Centrifuge at 200 3 g for 1 min and keep the flow-through.n.Repeat the previous wash, incubation, and centrifugation.o.Combine the flow-through with the first elution -these are your peptide samples.p. Transfer the peptide samples to MS sample vials and label them accordingly.

Note:
The peptide sample volume is approximately 100 mL.We recommend preparing pooled samples for quality control check and to improve the coverage for protein identification in the proteomic runs: for every 10 samples, take 9 mL from each sample and add to another MS vial labeled ''pool'' -you now will have 10 samples with 91 mL of sample and a pooled sample of 90 mL.q.Completely dry the peptide samples using vacuum centrifugation.
Note: Dried peptides are stable and can be kept at refrigerating temperature.

Sample analysis and data acquisition with mass spectrometry
Timing: 1-2 days (for step 9) In our study, the sample processing immediately prior to the analysis with high-resolution mass spectrometry was performed by the staff of the Biological Mass Spectrometry Facility (University of Manchester).Here we will describe the parameters used specifically for our study but can be adapted to other studies and/or instruments, accordingly.iii.18% B to 27% B over 8 min.iv.27% B to 60% B over 1 min.g.Wash the column at 60% B for 3 min before re-equilibration to 5% B in 1 min.h.At 85 min, increase the flow to 300 nL/min until the end of the run at 90 min.i. Use the Q Exactive HF hybrid operated in the HCD mode.j.Set up peptide selection for fragmentation automatically by data-dependent acquisition on a basis of the top 12 peptides with m/z ranging from 300 to 1750 Th, charge state of 2, 3, or 4, with dynamic exclusion set at 15 s.k.Set MS resolution to 120,000, with an AGC target of 3 6 , and a maximum fill time of 20 ms.l.Set MS2 resolution to 30,000, with an AGC target of 2 5 , maximum fill time of 45 ms, isolation window of 1.3 Th, and collision energy of 28.
Note: Parameters on the Q Exactive HF hybrid should be adjusted to different durations of runs.
Note: ECM fractions were run first, followed by cellular fractions after.Blanks were run in between fractions and time-point for the organoid analysis, to avoid carryover from one sample to another.

Processing and bioinformatics analysis of the proteomics data
Timing: 1 day (for step 10) Timing: 1-2 days (for step 11) 10. Day 5: Proteome Discoverer analysis of cellular and ECM sample fractions.We used the Thermo Scientific Proteome Discoverer software.The software has default qualitative workflows utilizing Sequest HT for peptide/protein identification, and quantitative workflow for label-free assays.
Note: Users can use other search engines and software like Andromeda and MaxQuant, Progenesis and Mascot, or use combined search engines integrated into Proteome Discoverer as we describe later.
CRITICAL: The cellular and ECM fractions are biologically distinct, i.e., their protein backgrounds are not comparable, and therefore, they should be analyzed separately.
a. Download and save the spectra data (.raw) to a local folder in your computer.b.Open Proteome Discoverer and create a new study.c.In the new study definition tab, add and assign adequate study factors corresponding to your assay conditions and replicates (this will impact the way the protein and peptide ratios are calculated by the software; the reader can refer to the software user guide for details about study factors for nested and non-nested assay designs).i. Add ''Individual'' factors to assign sample replicate.
ii. Add ''Categorical'' factors to assign experimental group or treatment.
d.In the ''Input Files'' tab, upload the .rawfiles for the analysis.e.In the ''Samples'' tab, assign to each .rawfile its respective category (group or treatment) and individual (sample replicate).
Note: In the ''Samples'' tab, keep all files assigned as ''sample'' in the ''Sample Type'' column.
Note: In the ''Precursor Ions Quantifier'' node, you can opt to change the scaling mode to none, and keep all the default settings for the rest of the workflow tree.
h. Select the ''Processing step'' in the ''Analysis'' window, and in the ''Workflows'' tab, open the ''PWF_QE_Precursor_Quan_and_LFQ_SequestHT_Percolator'' workflow from the ''Proces-singWF_Qexactive'' local folder -this is a default workflow for assays analyzed with the Q Exactive HF hybrid mass spectrometer.i. ''Select the ''Protein FDR Validator'' and check the default settings for target FDR (strict = 0.01, relaxed FDR = 0.05).Proteins that pass the strict threshold will be classified and high confidence match whilst those passing the relax (but not the strict) threshold will be classified and medium confidence matches.Low confidence matches should not be considered for further analyses.'' Note: Proteome Discoverer provides several default workflows for different instruments and analyses, with default settings.Select the adequate workflow accordingly.
Note: Users are encouraged to use the latest released versions of the target database and additional contaminant database of choice.j.In the ''Spectrum File RC'' and ''SequestHT'' nodes, set Protein database to Homo sapiens or Mus musculus (SwissProt and TrEMBL databases), Precursor Mass Tolerance = 10 ppm, Fragment Mass Tolerance = 0.02 Da, and Dynamic Modifications = Oxidation / +15.995Da (K, M, P), and leave the other setting as default.
CRITICAL: For this study, we used the SwissProt and TrEMBL databases (v.2018_01; OS = Mus musculus for mouse samples; OS = Homo sapiens for kidney organoids).

Protocol
[14] Note: By default, Proteome Discoverer uses Sequest HT but other search engine nodes are available and can be integrated into the workflow tree.For each extra search engine node, integrate a new ''Percolator'' node.k.From the ''Input Files'' tab, select the appropriate files, and drag and drop them into the ''Processing step'' box.l.In the ''Grouping and Quantification'' tab, select the appropriate study variables (i.e., groups or treatment), and for the bulk ratio generator, choose the appropriate group as a denominator for the calculation of protein ratios.
11. Label-free quantification and identification of extracellular matrix and basement membrane proteins a. Export the protein group files from Proteome Discoverer to an Excel file (.xls or .xlsx)and use this for the downstream analysis.b.To identify extracellular matrix and basement membrane proteins, download specie-specific ECM and ECM-associated protein lists from the matrisome project 15 (https://sites.google.com/uic.edu/matrisome/home) and basement membrane proteins from bmBASE 5 (https://bmbase.manchester.ac.uk/), and cross-reference with your data using gene symbols and/or UniProt identifiers.
Note: matrisomedb 16 and bmBASEdb 5 are online searchable databases that provide lists of individual matrix and basement membrane proteins, respectively, which are classified into different categories: collagens, adhesive glycoproteins, proteoglycans, regulators, matrixaffiliated proteins, secreted factors, cell surface interactors, and others.c.For the downstream analyses, consider only proteins assigned to a unique ''protein group'', quantified and identified by > 1 unique peptide, with a high/medium confidence match score (high: FDR < 1%, medium FDR > 1% and < 5% at protein level; use the same for peptide matches), and observed at least twice in each experimental group/condition.d.To calculate enrichment level for extracellular matrix and basement membrane proteins, first sum the intensity values (abundance) of all proteins in each respective category, then divide by the total sum of protein intensity to obtain the relative abundance of these protein categories per sample.
Note: Apply the same strategy to calculate the relative abundance of the different matrix and basement membrane protein categories.
e. Calculate the median extracellular matrix and basement membrane protein abundance per group and perform statistical comparisons with an ANOVA test.
Note: In our study, no missing value imputation was done, and the statistical analyses were carried out with Proteome Discoverer through its in-built two-way ANOVA test with post hoc Benjamini-Hochberg correction, with proteins with a p-value < 0.05 being considered as significant.
Note: If preferred, protein intensities can be log2-transformed prior to the abovementioned calculations to facilitate interpretation and visualization of the data.
Note: For pairwise comparisons, use a student's t-test or Welch's t-test, depending on group size, Gaussian distribution of the data, and variance disparity between groups.
f. Calculate fold changes for individual proteins and consider only those passing the statistical test with significant p-values (i.e., < 0.05).
Note: All box plots and bar plots for data visualization were obtained using the GraphPad Prism software.The Principal Component Analysis, Hierarchical Clustering, and heatmaps were obtained using RStudio and the ggplot2 package. 10. Data integration.In this step, we reprocessed proteomic data from human glomeruli and kidney tubulointerstitium (PRIDE: PXD026002, PXD022219) with Proteome Discoverer and further compared with human kidney organoid and mouse fetal kidney data with Spearman's rank correlation.a.To calculate the correlation coefficient and generate correlation plots, perform analyses for the cellular and ECM fractions separately, using the ComplexHeatmap package 11 for R. 13.Gene ontology.
a. Prepare a list of differentially expressed proteins (for example 2-times fold-change, p-value < 0.05 or FDR < 0.05; or up-and/or down-regulated) for the gene ontology (GO) analysis.b.Prepare a second list of all proteins detected in your system (kidney organoids, mouse kidneys) for use as the protein background for GO.
Note: In our study, we used DAVID Bioinformatics Resources 17 or GO.Readers can find a detailed tutorial at https://david.ncifcrf.gov/helps/tutorial.pdf.
Step 1: upload the protein background, the list of proteins differentially expressed, and select the adequate identifier (e.g., ''OFFICIAL_GENE_SYMBOL'' if using gene names).
Note: When using gene names, indicate the adequate species for the analysis (e.g., Homo sapiens for the kidney organoids, Mus musculus for the mouse kidney).
e. Submit the lists.f.Step 2: for the functional annotation tools, select the desired levels for GO annotation.
Note: In our study, we searched for Gene_Ontology ''DIRECT'' terms, and ''FAT'' for more specific terms.
g. Click on ''Functional Annotation Chart'' to generate a GO report with GO terms and adjusted statistical significance (Benjamini-Hochberg).
Note: In our study, we used the STRING database 19 (https://www.string-db.org/) and Cytoscape 8 to generate and customize protein interactomes.The reader can find detailed STRING tutorials at https://www.string-db.org/cgi/help/.
a. Go to STRING and search interactions for a list of multiple protein candidates.b.Set a minimum interaction score of > 70% to obtain high-confidence interactions.
c. Export the data as a short tabular text output to upload to Cytoscape.d.Generate and customize a protein interactome using the data from STRING.

EXPECTED OUTCOMES
To date, several ECM enrichment and purification strategies and processing approaches have been developed and successfully applied by other groups to unravel the molecular complexity and structure of ECM in different biological 14,[22][23][24][25][26][27][28][29][30] Our strategy (Figure 2) mostly relies of differential solubility of ECM and ECM-associated proteins, and with it we have defined the global composition of basement membranes in cell-based and animal tissue systems, and specifically elucidated molecular changes throughout organoid differentiation (Figure 3).This strategy is also expected to reveal the composition of interstitial matrices and changes in diverse contexts, such as development and disease.By fractionating the samples used in our study, we could identify and quantify over 6,600 proteins in kidney organoids, and over 5,000 in fetal mouse kidneys. 1 The depth of coverage for identification and quantification of matrix and basement proteins with our enrichment strategy is expected to be higher than for assays using whole tissue lysates (Figure 3), but can also depend on several factors mentioned in this protocol, such as sample amount for fractionation, the inclusion of post-translational modifications for protein identification, the efficiency of trypsin digestion, the performance of the mass spectrometer, and the use of data-dependent acquisition (DDA) versus data-independent acquisition (DIA) modes, etc.

LIMITATIONS
An important limitation in our approach the absence of effective tissue compartmentalization during the processing of tissue fractions.However, this limitation can be addressed through the isolation of distinct tissue compartments.For instance, differential sieving or laser microdissection can be employed to separate kidney glomeruli and tubules.This isolation step may result in a substantial reduction in the initial quantity of tissue samples available, which in turn could affect the yield of fractionation and pose challenges in achieving a comprehensive proteome coverage.This is particularly applicable when dealing with low-abundance or insoluble matrix proteins, such as basement membrane collagens.

TROUBLESHOOTING
Problem 1 ECM-enriched pellet may be difficult to solubilize during the fractionation (step 1o).

Potential solution
In this case, we recommend homogenizing the samples properly prior to the fractionation incubations, using a sterile blade and a syringe equipped with a gauge needle.Optimizing the sonication parameters may facilitate the dissolution of ECM-enriched pellet, and repeated rounds of sonication may be necessary.Furthermore, consecutive solubilization and pelleting down samples may be beneficial; however, this may increase the number of fractions, which will increase the cost for analysis.

Potential solution
The suggested trypsin-to-sample ratio of 1:10 should efficiently digest the recommended protein concentration.In case of incomplete digestion, it may be beneficial to conduct multiple rounds of trypsin digestion, either at 37 C for 12 h or at 47 C for 60 min.Alternatively, consider multi-enzyme digestion, such as using trypsin with LysC in conjunction with collagenase. 31It is important to note that opting for multi-enzyme digestion will have implications for the subsequent bioinformatics analysis, and this consideration should be factored into your experimental planning.

Problem 3
Results show lower protein identification coverage (step 11).

Potential solution
The success of the fractionation relies partially on having enough tissue samples.Pooling samples prior to the enrichment step may be considered to increase the amount of starting material to later improve the number of proteins identified in the proteomic analysis.The user should consider individual and batch-to-batch variations when interpreting the results.Alternative approaches to enhance ECM protein identification may include deglycosylation of tissue lysates with glycosidases. 32[35][36] Problem 4 Results show multiple entries for the same protein or protein group (step 11).

Potential solution
This often happens due to multiple spectra/peptides that match the same protein or protein group.We recommend reviewing the protein sequence database used in the search.Use the most up-todate version of the database and ensure that it only includes relevant/unique protein entries to minimize redundancy of the results.Ensure that you are filtering out low-confident peptides, and those that are not unique and confidently matched to specific proteins.Additionally, avoiding the use of databases that include multiple entries for different isoforms or protein fragments, such as TrEMBL Fasta files, can help resolve ambiguities in the results.

Problem 5
Batch-to-batch and inter-batch variations for the kidney organoids (step 10f).

Potential solution
Due to batch-to-batch variation of kidney organoid differentiation driven by differences in the rates of organoid maturation, we recommend processing organoids from the same batch to achieve comparable datasets.We also suggest the practice of pooling samples to reduce inter-batch variations.
On the other hand, batch-to-batch variation could help identify robust signatures/differences.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Rachel Lennon (Rachel.Lennon@manchester.ac.uk).

Materials availability
This study did not generate any new unique reagents and/or materials.STAR Protocols 4, 102741, December 15, 2023 Protocol

3. Day 1 :
Reduction and alkylation.a. Place 50 mL or 100 mL of sample fraction into clean sample tubes.b.Add 2.5 mL or 5 mL of 100 mM DTT stock solution to 50 mL or 100 mL of sample fraction, respectively, to a final concentration of 5 mM of DTT, and incubate for 10 min at 60 C using a heating block to reduce proteins.c.Let the samples cool down to 20 C then add 7.5 mL or 15 mL of 100 mM IAM stock solution to 50 mL or 100 mL of sample fraction, respectively, to a final concentration of 15 mM of IAM, and incubate for 30 min at room temperature, in the dark, to alkylate proteins.d.Add the same amount of DTT as previously to quench the residual alkylation reaction, and vortex mix briefly.4. Day 1: Protein quantitation.

6. Day 2 :
Trypsin digestion.a. Move the spin columns to clean 1.5 mL collecting tubes.b.Prepare 20 mg of Sequencing Grade Modified Trypsin (Promega) in 200 mL of digestion buffer.

8 .
Day 3: Peptide desalting, cleaning, and drying.a. Prepare the OLIGO R3 beads in 50% ACN in a glass vial and let them settle into the bottle of the vial.b.Add 10 mL of the settled OLIGO R3 beads into the appropriate number of wells of a Corning FiltrEX 96-well filter plate equipped on top of a 96-well collecting plastic plate.c.Wash the beads with 200 mL of 50% ACN.d.Centrifuge the plate at 200 3 g for 1 min to remove the liquid.Note: Discard the liquid containing ACN to an adequate non-chlorinated solvent waste container.

Figure 2 .
Figure 2. Sample acquisition and workflow for sample fractionation and processing for trypsin digestion for mass spectrometry analysis Created with BioRender.comwith publication license agreement number: HD25XU5O15.

Figure 3 .
Figure 3.The expected level of enrichment for matrix and basement membrane proteins during human kidney organoid differentiation The plot on the left shows the relative abundance of matrix proteins identified and quantified in the cellular and ECM fractions of kidney organoids at days 14, 18, and 25 of differentiation, through our fractionation strategy; on the right, the plot indicates the crescent level of basement proteins quantified in the kidney time course study.Pooled data are presented as median, and error bars indicate the 95% confidence interval for the median.Adapted from Morais, Tian et al. under a CC BY 4.0 Attribution license [creativecommons.org]with permission.
This solution should be prepared freshly and kept on ice until use.Add 1 tablet of EDTA-free protease inhibitor cocktail and vortex to dissolve right before use.
This solution should be prepared freshly and kept on ice until use.Add 0.04 mL of 5 M NH 4 OH right before using.Stable at 20 C for long-term storage (several weeks).Adjust to pH 7.5.Use LC-MS grade water to dilute to 13.