Organoid Sample Preparation and Extraction for LC-MS Peptidomics

Summary This protocol describes the peptidomic analysis of organoid lysates, FACS-purified cell populations, and 2D culture secretions by liquid chromatography mass spectrometry (LC-MS). Currently, most peptides are quantified by ELISA, limiting the peptides that can be studied. However, an LC-MS-based approach allows more peptides to be monitored. Our group has previously used LC-MS for tissue peptidomics and secretion of enteroendocrine peptides from primary culture. Now, we extend the use to organoid models. For complete details on the use and execution of this protocol, please refer to Goldspink et al. (2020).

CRITICAL: All organoid culture should be carried out under aseptic conditions in a biosafety hood.
CRITICAL: The use of human or animal tissue for research purposes is subject to ethical and legislative restrictions, appropriate approvals must be obtained before commencing work.

MATERIALS AND EQUIPMENT
Note: Saline buffer can be stored at 2 C-8 C for up to 6 months after preparation. 0.001% (w/ v) fatty acid-free BSA and glucose (typically 10 mM) are added on day of use.
Note: FACS media (without Y-27632) can be stored at 4 C for up to 1 month. Y-27632 should be added on day of use. Note: 6 M guanidine hydrochloride can be stored at 22 C for up to 3 months after preparation.
Note: 50 mM ammonium bicarbonate (aq) can be stored at 22 C for up to 3 months after preparation.
Note: This solution is prepared fresh immediately before use.
Note: This solution is prepared fresh immediately before use.

LC-MS System Setup
Samples are loaded onto a 0.3 3 5 mm peptide trap column (Thermo Fisher Scientific) at a flow rate of 30 mL/min and analyzed using a 250 3 0.075 mm Easy-Spray column (Thermo Fisher Scientific with skin. Formic acid is highly corrosive to eyes, skin, and the respiratory system and should therefore always be added in a fume hood. Eluted peptides are directly introduced into the mass spectrometer by positive nano electro-spray ionization using a spray voltage of 1.8 kV, and an S-lens setting of 70 V. The mass spectrometer is set to perform full MS scans at 1 microscan with a resolution of 70,000, over a mass range of 400-1,600 m/z. Following each survey scan, the top 10 ions of each spectrum are selected for MS/MS analysis, and ions selected for fragmentation are added to an exclusion list for 60 s (Table 2).
Alternatives: This protocol is illustrative of our specific LC-MS/MS approach using a Thermo Fisher Scientific Ultimate 3000 nano LC system coupled to a Q Exactive Plus Orbitrap mass spectrometer. However, alternative LC-MS systems with similar capabilities (nanoflow, mass accuracy, resolving power and m/z range) should be suitable. Note: Using ice-cold Cell Recovery Solution and PBS aids with the removal of organoids from extracellular matrix (of the BME domes) and also reduces peptide degradation.
CRITICAL: Avoid touching organoids while removing supernatant as they will readily stick to pipette tips. BSA should not be added above 0.001% to prevent sticking as this will interfere with the LC-MS analysis.
CRITICAL: Remaining matrix may interfere with peptide extraction and LC-MS. Organoids should be washed with ice-cold PBS until no visible matrix remains (normally 2-3 times).
3. Add 25 mL of 6 M guanidine hydrochloride per organoid dome to resuspend pellet (typically 300-500 mL is used per confluent 6-well) and snap freeze on dry ice.
Pause Point: Samples can be frozen at À70 C until ready to continue with peptide extraction

Peptide Extraction of Lysed Organoids
Timing: 21 h This method describes peptide extraction of whole lysed organoid cultures.
4. Defrost the samples on ice and precipitate with a 1:5 ratio of 80% acetonitrile (ACN) in water. a. If necessary, transfer samples to a larger Eppendorf tube prior to precipitation.

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Note: Precipitation will result in the formation of three layers within the sample; organic, aqueous, and solid cell precipitate. Centrifugation and placing the sample on ice will clarify the layers, see step 5.
5. Centrifuge at 3,500 3 g for 5 min at 4 C and transfer the aqueous phase (lower), without disturbing the precipitate, to a clean LoBind Eppendorf tube. 6. Evaporate the supernatant in a rotary evaporator until dry (usually takes~18 h; overnight evaporation). 7. Reconstitute in 250 mL 0.1% formic acid in water (v/v) and transfer onto an Oasis HLB Prime mElution solid phase extraction (SPE) plate (Waters) and gently apply positive pressure on an SPE manifold. 8. Wash all samples with the following reagents, ensuring the SPE plate reservoirs are clear before each step. a. 200 mL 0.1% formic acid in water (v/v). b. 200 mL 5% methanol:1% acetic acid in water (v/v). 9. Elute the samples into a clean QuanRecovery plate with two 30 mL aliquots of 60% methanol:10% acetic acid in water (v/v). 10. Evaporate the supernatant, under nitrogen at 40 C on a Biotage SPE Dry 96 system until dry. 11. Reconstitute into 75 mL 10 mM DTT in 50 mM ammonium bicarbonate. 12. Incubate the samples for 1 h at 60 C. 13. Add 20 mL 100 mM iodoacetamide in 50 mM ammonium bicarbonate. 14. Incubate for 30 min at 18 C-25 C, in the dark. 15. Dilute the samples with 25 mL 1% formic acid in water (v/v).
Note: Reduction and alkylation (steps 11-14) breaks disulfide-bonded peptides so they can be identified in the bioinformatics analysis. These steps can be removed but this will reduce the number of positive peptide matches. If a targeted quantitation approach is being used, the SPE extract is to be diluted with 75 mL 0.1 % formic acid in water (v/v) after step 9 for LC-MS/MS analysis. Note: Digestion to single cells typically requires 30-60 min incubation with TrypLE at 37⁰C, depending on organoid size and morphology. To enhance digestion, organoids can be transferred to a 50 mL tube during step 17 (which prevents organoids settling to the bottom of the tube) but should be returned to 15 mL tubes for centrifugation. Some small clusters of up to ten cells are acceptable at this stage as they will be further broken up by mechanical trituration.

Purification of Cell Populations by FACS
18. Wash organoids with FACS media. a. Centrifuge at 400 3 g for 5 min to pellet single cells. b. Discard supernatant. c. Mechanically triturate organoids 10-20 times each with P200 and P20 micropipettes. d. Resuspend pellet in 5 mL of FACS media. e. Repeat steps a-d two more times, then pass cell suspension though a 50 mm cell strainer. 19. Repeat steps 18a-c and resuspend in FACS media supplemented with live cell dyes (for example, 5 mM DRAQ5 and/or 2 mg/mL DAPI). 20. Transfer single-cell suspension to 5 mL round bottomed polypropylene FACS tubes. 21. Sort cell populations of interest directly into 350 mL of 6 M guanidine hydrochloride in LoBind 1.5 mL Eppendorf tubes.
Note: Where possible we recommend collecting >10,000 cells per population for peptidomic analysis, although we have previously measured high-abundance peptides from as few as 100 cells from fresh tissue. Follow manufacturer instructions regarding use of cell sorters. See Figure 1 for an example FACS sort layout. Pause Point: Samples can be frozen at À70 C until ready to continue with peptide extraction Note: As there is 10% FBS present in the FACS media, some bovine derived peptides will be carried through to LC-MS analysis. The impact on peptidomics is minimal, however for proteomic analysis the bovine peptides will generate interferences to the database search. If interested in proteomics analysis, use 0.1% BSA or a lower concentration of FBS in FACS media.

Extraction of FAC-Sorted Cells
Timing: 21 h 23. Thaw sorted cell samples on ice and then re-freeze on dry ice (or in a À70 C freezer) followed by defrosting on ice. 24. Add 900 mL of 80% ACN in water to each sample and vortex mix. 25. Centrifuge at 12,000 3 g for 5 min at 4 C and transfer the aqueous phase (lower) to a clean Lo-Bind Eppendorf. 26. Evaporate the supernatant in a rotary evaporator until dry (usually takes~18 h; overnight evaporation). 27. Reconstitute in 500 mL 0.1% formic acid in water (v/v). 28. Transfer samples onto an Oasis HLB Prime mElution solid phase extraction (SPE) plate (Waters) and gently apply positive pressure on an SPE manifold.

Generation of 2D Organoid Cultures
Timing: 20 h This method describes the preparation of 2D organoid cultures for secretion experiments. The number of organoids required will depend on expression levels of the peptides of interest and differentiation conditions. To assess gut hormone secretion from intestinal organoid cultures (as described in Goldspink et al., 2020), we recommend that one confluent well of a 6-well plate is used to seed six wells of a 24-well plate. a. Aspirate organoid culture media. b. Add 2 mL of ice-cold Advanced DMEM/F12 (ADF) per 6-well. c. Use a P1000 micropipette to scrape and collect organoids into a 15 mL centrifuge tube. d. Centrifuge at 500 3 g for 5 min to pellet organoids then discard supernatant. 39. Resuspend organoid pellet in 4 mL of TrypLE Express per 6-well.
a. Incubate in a water bath for 8-12 min at 37 C. b. Centrifuge at 400 3 g for 5 min to pellet organoids then discard supernatant. c. Triturate 10-20 times with a P200 micropipette to mechanically disrupt the organoid pellet.
Note: For optimal attachment, aim for small clusters of 10-20 cells (this can be visualized after plating or by directly observing cells in a 15 mL tube at any point under a light microscope).
a. Immediately add pre-warmed organoid culture media + 10 mM Y-27632 (250 mL/24-well) to each well. 42. Plate 250 mL of the cell suspension per well.
Note: To ensure consistency between wells, mix the cell suspension by pipetting regularly during plating to ensure the suspension remains homogenous. After plating, gently shake plates (left to right and back to front) to ensure cells do not clump in the center of the well.

Collection of 2D Secretion Supernatants
Timing: 2 h This method describes the collection of supernatants from 2D organoid cultures stimulated under different conditions. This is performed in 24-well plates and it may be necessary to combine supernatants collected from multiple wells, depending on the peptides of interest and LC-MS sensitivity.
To measure ileal gut hormone secretion (as described in Goldspink et al., 2020), we combine supernatants from three wells of a 24-well plate. Strong stimulants -such as forskolin/IBMX (each 10 mM) which elevate intracellular cAMP levels -can be used to assess secretion of peptides from a range of cell types. Alternatively, drugs which target specific receptors or molecular pathways can be tested to determine whether these stimulate or inhibit secretion of peptides of interest.

Following overnight incubation, observe cultures under a light microscope (Figure 2)
Note: See Expected Outcomes and Troubleshooting 2 45. Add 0.001% fatty acid-free BSA and, if required, glucose (normally 10 mM) to an appropriate volume of saline buffer and pre-warm to 37 C (this solution is termed secretion buffer). 46. Wash cultures with pre-warmed secretion buffer.
a. Aspirate overnight culture media. b. Add approximately 300 mL of warm secretion buffer to each well to wash cultures. c. Aspirate secretion buffer. d. Repeat steps b-c twice more then add a further 300 mL of warm secretion buffer to each well and incubate at 37 C for 30 min (meanwhile proceed to step 47).
Note: These wash steps should not be too gentle as they aim to remove unattached cells or debris and wash off all culture media (which may interfere with LC-MS). The 30 min pre-incubation step allows acclimatization of cells to the low-nutrient secretion buffer.
47. Prepare stimulant solutions in secretion buffer (200 mL is required per well of a 24-well plate, and should be made up with 10% excess volume) and keep at 37 C. 48. After the 30 min pre-incubation, remove all the secretion buffer from wells (using a P1000 micropipette with the plate tilted to a 45 angle) and add 200 mL of stimulant solution to each well.
Note: To avoid cells drying out, buffer should only be removed from 3-4 wells at a time 49. Incubate with stimulants at 37 C for 1 h. a. Meanwhile label and pre-chill (on ice) two LoBind 1.5 mL Eppendorf tubes per condition. 50. Collect supernatants into pre-chilled LoBind 1.5 mL Eppendorf tubes.
Note: Supernatants from multiple wells can be combined at this step, if required for peptide detection by LC-MS. Steps 50-52 should be performed on ice using pre-chilled tubes to minimize peptide degradation 51. Centrifuge at 2,000 3 g for 5 min at 4 C to pellet any debris or detached cells. 52. Transfer 180 mL (or greater volume if multiple wells were combined in step 50) of each supernatant to fresh LoBind tubes, taking care to avoid touching any pellet which forms.
CRITICAL: As intracellular levels of secreted peptides are normally higher than in supernatants, any detached cells will interfere with quantification 53. Immediately snap freeze on dry ice.
Pause Point: Samples can be frozen at À70 C until continuing with peptide extraction

Extraction of Secretion Samples
Timing: 1 h acetic acid in water (v/v). 59. Dilute the samples with 75 mL 0.1% formic acid in water (v/v).

Analysis of Peptides by Liquid Chromatography Mass Spectrometry
Timing: 130 min per sample This step describes sample data acquisition of extracted samples by LC-MS analysis. Extracts are analyzed using a Thermo Fisher Scientific Ultimate 3000 nano LC system coupled to a Q Exactive Plus Orbitrap mass spectrometer according to the analytical parameters stated previously in LC-MS system set up.
60. Prior to injection onto the liquid chromatography (LC) system, centrifuge samples at 3,500 3 g for 10 min. 61. Inject samples onto LC-MS system for analysis; recommended volumes of 30 mL for secretion supernatants and lysed organoids, and 40 mL for sorted cells.

Quantification of Peptides in Raw LC-MS Data Files Using Qual Browser and Quan Browser Software
Timing: up to 4 h depending on the number of LC-MS data files being searched ll OPEN ACCESS 62. Using PEAKS software (v8.5), open a representative LC-MS file in Qual Browser that will contain peptides of interest. 63. Extract the m/z ion specific for the peptide (as specified in the PEAKS software viewer) using the ranges function on the chromatogram pane ( Figure 3B). 64. Sum spectra under the peak at the expected retention time as specified in the PEAKS software ( Figure 3C). 65. Note the m/z values of the peptide 13 C isotopes for the peptide, usually the top 4 ions will suffice. Adding ions of low abundance can sometimes have little impact on signal, but can increase chromatogram noise.  Figure 4. 68. Open file list containing all LC-MS files and select batch reanalysis function to perform quantitative analysis of the data using the specified processing method. 69. Check integration of peptide peaks are correct, and if needed make changes to integration manually, or update the automatic integration parameters to reprocess all the integrations for a specific peptide ( Figure 5).

EXPECTED OUTCOMES
Each sample injected onto the LC-MS system will generate a Total Ion Chromatogram (TIC) which is made up of all ionized species and can be filtered by mass ranges to identify the presence of specific peptide fragments. The data for each sample are processed using PEAKS software (v8.5) which matches experimentally acquired MS/MS spectra against proteins in the UniProt database ( Figure  3A). Exporting data from the PEAKS software generates relative quantitative data at the protein level such that comparison between samples can be performed in other statistical software packages, such as RStudio. See Figure 3 for an example of a peptide m/z isotope pattern and the identification of the specific retention time for peptide peak area integration purposes. This approach allows for the generation of semi-quantitative measurements of peptides -or fully quantitative if peptide standards are co-extracted and analyzed.

QUANTIFICATION AND STATISTICAL ANALYSIS
To compare the levels of specific peptides, it is recommended that the raw data are interrogated manually. Each peptide generates a signature pattern in the MS data, and will acquire a varied number of charges to generate predictable mass to charge ratio (m/z) values. Close interrogation of each charge state will show that peptides will regularly have multiple 13 C isotopes, therefore it is important to identify these and include them in the quantitative analysis to improve sensitivity.
Software packages that are supplied with the instrument, such as Qual Browser (Supplied in Xcalibur), can be used to interrogate the raw data to identify the specific m/z values for targeted peptide quantitation. Once the specific m/z patterns for each target peptide are identified, these can be used to quantify multiple peptides using other software packages, such as Quan Browser (Supplied in Xcalibur). Once the retention time of the peptide and the m/z values are identified, then the peak integration can be optimized for each peptide to ensure reproducibility in peak area assignment. Figures 4 and 5 shows an example of sample quantitation from Goldspink et al. (2020).
Peak area values can then be compared between samples in statistical software, such as RStudio.

LIMITATIONS
Due to the chemical properties of peptides, not all peptides are extracted equally by this method and the extraction recovery and sensitivity of the LC-MS approach may vary for different peptides.
Nanoflow liquid chromatography runs take approximately 2 h to perform. This makes the method low throughput and therefore not suitable for large sample sets. The approach is also relatively expensive and requires the use of high cost and complex instrumentation.
The full scan peptidomic method described generates significant amounts of data (approximately 1 GB per 2 h analysis), which can be time consuming to process. Additionally, this type of measurement will identify all ionized species in the samples which may include unwanted contaminants, such as BSA, media components, and peptides from typical cell turnover processes. These may compete for MS/MS fragmentation selection with peptides of interest, which will reduce the depth of peptide identification in samples. If data are only required on known peptides, a more targeted analysis using a triple quadrupole MS system can be used instead of a high-resolution mass spectrometer. These systems are specifically designed to be used for quantitative peptidomics analysis and are usually employed in a higher throughput manner compared to traditional discovery peptidomics methods.

Potential Solution
Over time, the efficiency of LC-MS consumables (columns and traps) can decline, this leads to poor chromatographic separation, shifts in retention time, and broadening of peptides which results in a reduction in the sensitivity of the LC-MS.
Regular checks on the suitability of the LC-MS system should be performed by analyzing a standard peptide solution and monitoring the peak retention times and signal of key peptides. Trap column cartridges can be replaced quickly, while new analytical columns require more effort to get running again.
Over time the MS instrument might accumulate dirt on the orifice, reducing sensitivity which will require the transfer tube to be removed and cleaned.

Potential Solution
When plating 2D cultures, organoids should be broken up into small clusters of 10-20 cells. If organoids are not sufficiently broken up, then 3D structures may reform during overnight incubation. By contrast, if organoids are broken down too harshly, they form sparse or single-cell cultures. Ensure Y-27632 is added to culture media during plating to limit anoikis-induced cell death. Plating density must be optimized based on organoid size and number of organoids per well. Some differentiation-promoting compounds (such as Notch inhibitors) may also interfere with cellular attachment. Cultures should be plated on a thin coat of Matrigel (2%) to improve attachment. Increasing the Matrigel pre-coating period from 1 h to up to 8 h may improve poor cellular attachment. See Figure 2for a comparison of successful and unsuccessful organoid cultures.

Problem 3
Expected peptides not detected by LC-MS (step 61).

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Potential Solution This could be caused by poorly differentiated organoids lacking mature cell types. Organoid culture protocols should be optimized prior to peptidomic analyses using techniques such as qPCR, immunostaining, and the presence of fluorescent reporters. Methods used for culture of human small intestinal organoids are described in Goldspink et al, 2020.
LC-MS analyses are generally less sensitive than antibody-based methods and therefore a large number of organoids are normally required for sample extraction, depending on the expression levels of the peptides of interest. We recommend analyzing organoid lysates by LC-MS/MS to make sure they are producing the peptide of interest before attempting experiments using sorted cells or secretion supernatants. Detection of gut hormone peptides can be improved by using culture protocols which selectively drive the enteroendocrine lineage (Beumer et al., 2020, Goldspink et al., 2020.
In some instances, peptides may not be found during database searches which can be caused by peptide degradation. The presence of breakdown products can be used to confirm if this has occurred during sample extraction. The use of short incubation periods and performing sample collection on ice is more effective at preventing over-fragmentation than the addition of protease inhibiting peptides. The addition of formic acid immediately prior to extraction after thawing will help reduce proteolytic activity, while the addition of 6 M GuHCl to the homogenate analysis will also prevent degradation during extraction.

RESOURCE AVAILABILITY Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Richard Kay (rgk27@medschl.cam.ac.uk).

Materials Availability
No new materials were generated in this study.

Data and Code Availability
The sorted cell peptidomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (PXD017825).