Materials and Reagents

Materials

Conical tubes, 50 mL (Biologix, catalog number: 10-9502)
Polypropylene centrifuge bottle with Screw-On Cap, 250 mL (Beckman Coulter, catalog number: 356011)
Polypropylene centrifuge bottle with Screw-On Cap, 50 mL (Beckman Coulter, catalog number: 357003)
Metal spatula
Glass beads 0.5 mm diameter (Millipore Sigma, Sigma, catalog number: G8772)
Glass tubes (Kimax, catalog number: 45048)
Glass eyedropper
Chromatography paper sheets (Sartorius Stedim FN3 580 × 600 mm)
Scintillation 20 mL glass counting vials (Wheaton, catalog number: 986532)

Reagents

Yeast extract (Bacto^TM, catalog number: 212750), store at room temperature (RT)
Adenine (Millipore Sigma, Sigma, catalog number: A8626), store at RT
Yeast nitrogen base (YNB) (Thermo Fisher Scientific, BD Difco, catalog number: DF0392-15-9), store at RT
Puromycin 10 mg/mL (InvivoGen, catalog number: ant-pr-1), store at 4 °C
Cycloheximide 2 mg/mL (Millipore Sigma, catalog number: C-6255), store at -20 °C
D-[¹⁴C(U)]-Glc (Perkin Elmer, catalog number: NEC042V20UC), store at 4 °C
Glucose 1 M (Dextrose 1-hydrate, Biopack, catalog number: 9812.08), store at RT
Methanol 99.9% (Millipore Sigma, Merck, catalog number: 106009), store at RT
Chloroform (Merck, catalog number: 107024), store at RT
1-propanol 98% (Millipore Sigma, Merk, catalog number: 100997), store at RT
Nitromethane (Millipore Sigma, catalog number: 270423), store at RT
Hydrochloric acid fuming 37% for analysis (Merck, catalog number: 1003171000)
PPO-POPOP scintillation cocktail (concentrated scintillator PPO-POPOP, RPI, catalog number: 111045)
Milli Q water
Yeast extract medium supplemented with adenine (YEA) (see Recipes)
Yeast nitrogen base (YNB) without glucose (see Recipes)
Radioactive solution to label a 500 μL sample (see Recipes)
Chloroform:methanol:water (1:1:0.3) (see Recipes)
Chloroform:methanol:water (1:16:16) (see Recipes)
0.02 N HCl (see Recipes)

Equipment

1 L glass flask
Orbital shaker (for flasks) set at 28 °C (Forma Scientific, model: 4581)
OD₆₀₀ reader (Amersham Biosciences, model: Ultraspec 10)
Centrifuge (Beckman Avanti) with rotors JA-14 and JA-20
Thermomixer set at 28 °C (Thermo-shaker, mrc)
Water bath (Vicking SRL)
Airflow (Aquarium air pump RS-Electric, model: RS-15000)
Analog benchtop centrifuge (Rolco)
Vortex (Scientific Industries, model: Vortex-genie 2)
Freezer -20 °C (KENT)
Glass chromatography chamber (Homemade)
Scintillation counter (Pharmacia, RackBeta 1214/1219)
NOTE: The equipment described here may easily be replaced for common lab equipment of other brands.

Software

Microsoft Excel (version 2016) (Microsoft.com)

Procedure

In vivo labeling of LLOs

Harvesting S. pombe cells (4 h)
1. Inoculate a 50 mL Falcon tube containing 5 mL of sterile YEA medium with an isolated colony of the desired S. pombe strain. Grow overnight at 28 °C and 250 rpm in an orbital shaker.
2. The following morning inoculate 250 mL of sterile YEA in a 1 L glass flask with 1 mL of the overnight S. pombe culture.
3. Grow at 28 °C and 250 rpm until reaching the exponential growth phase (OD = 1–1.5). The time may vary depending on the strain.
4. Centrifuge 200 mL of the grown culture in 250 mL centrifuge bottles at 3,000 × g and 4 °C for 5 min.
5. Discard the supernatant.
6. Resuspend the pellet in 30 mL of cold 1% YNB. In this step, it is convenient to first resuspend the cells in 10 mL of ice-cold YNB using a 10 mL glass pipette and a pipette aid, and then add the remaining 20 mL.
7. Transfer the 30 mL sample to a 50 mL centrifuge bottle tube previously weighed. Keep it on ice. Weighing the tubes will be important later to calculate the wet mass of cells.
8. Centrifuge at 3,000 × g and 4 °C for 5 min and discard the supernatant.
9. Resuspend the pellet in 30 mL of YNB as described in step A6 and centrifuge as in step A8.
10. Discard the supernatant. Remove the remaining drops by placing the tube face down over a tissue paper.
11. Wash the pellet again with YNB by repeating steps A9–A10.
12. Weigh the tube with the pellet and calculate the wet mass of cells by subtracting the weight of each empty tube. Add two volumes of cold YNB per gram of cells (v/w). If you have 0.5 g of wet weight, you should add 1 mL of media. Resuspend cells with a pipette.
13. Take 500 μL of the cell suspension and place it into a new 50 mL centrifuge bottle, where the labeling will be performed.
Preparing reagents for radioactive pulse (15–30 min)
1. Add 200 μCi of [¹⁴C]-glucose per sample in a glass tube within a chemical hood and dry by exposure to a constant airflow at room temperature. Follow the approved procedures to manipulate radioactive liquids in your institution.
2. Once dried, solubilize the radioactive material in 50 μL of YNB containing 50 mM Glc (see Recipe 3).
Yeast cells labeling (20 min)
1. Add the protein synthesis inhibitors puromycin and cycloheximide to the 500 μL cell suspension from step A13 to reach final concentrations of 50 μg/mL and 70 μg/mL, respectively. Incubate tubes for 2 min at 28 °C.
2. Add the entire radioactive solution to the cell suspension. Incubate the sample at 28 °C for 15 min at 200 rpm using a thermomixer.
3. Stop the pulse by transferring all the cells to a glass tube containing 5 mL methanol.
Extraction of LLOs
Notes:
1. This step will take 6 h the first day and 4 h the next day if the protocol is interrupted at step 19; alternatively, 10 h if it is entirely performed on the same day.
2. Extraction of LLOs follows the following rationale: these compounds have both large hydrophilic and hydrophobic moieties. Thus, they are insoluble in solvents as chloroform:methanol (3:2), which is poorly hydrophilic but able to solubilize phospholipids, or in methanol or water, which are poorly hydrophobic but able to solubilize water-soluble metabolites. On the contrary, chloroform:methanol:water (1:1:0.3) is a single-phase solvent that displays both hydrophilic and hydrophobic features and thus is able to extract dolichol-linked oligosaccharides.
1. Centrifuge the glass tube at 2,000 × g for 3 min at room temperature and discard the supernatant carefully.
  Notes:
  1. All the following centrifugation steps of Procedure D should be performed under these same conditions.
  2. Follow institutional rules to discard radioactive material.
2. Add 5 mL of chloroform:methanol (3:2) and resuspend the pellet completely by vortexing. This pellet is particularly tough to resuspend. To do so, it is convenient to place a small spatula in the tube at the moment of vortexing. The spatula must be actively kept inside the tube during the shaking of the sample. Remove the spatula from the tube after the pellet has been resuspended and leave it in a large beaker containing water until next use.
3. Repeat steps D1–D2 twice, completing three total extractions with chloroform:methanol (3:2).
4. Add 5 mL of methanol and resuspend the pellet as in step D2.
5. Centrifuge and discard the supernatant.
6. Finally, add 5 mL of Milli Q water and resuspend the pellet by vortexing, using the spatula to help.
7. Centrifuge and discard the supernatant.
8. Resuspend cells in 2 mL of ice-cold water and 0.5–1 mL of glass beads.
9. Vortex the sample for 10 min. Stop the vortexing every 1 min to put the sample on ice for 1 min and thus avoiding overheating.
10. Centrifuge.
11. Add Milli Q water to complete a volume of 5 mL.
12. Centrifuge and carefully discard the supernatant using an eyedropper.
13. Repeat four more times the water extraction steps D11–D12.
14. Add 5 mL of chloroform:methanol:water (1:1:0.3) and resuspend the pellet vigorously by vortexing.
15. Centrifuge.
  Note: After centrifugation, it is common to see two phases in the supernatant instead of one. If this is the case, use an eyedropper to carefully add methanol drop by drop until observing just one phase.
16. Collect the supernatant in a new glass tube and start drying the sample using an airflow at room temperature. It is possible to accelerate the process by drying the supernatant at higher temperatures, placing the tube in a water bath under the air flow, without exceeding 40 °C (see Figure 2).
  
  Figure 2. Schematic representation of the water bath and the adapted airflow used for drying samples. A commercial air pump for fish tanks was employed to supply airflow.
17. In parallel, add 5 mL of chloroform:methanol:water (1:1:0.3) to the remaining pellet obtained in step D15 and mix it by vortexing. Centrifuge.
18. Collect this new supernatant using an eyedropper and pool it with the supernatant obtained in step D15 while it is being dried.
19. Repeat steps D16–D17 three more times. At the end of this extraction step, all the collected supernatants from a single sample will be pooled and dried in the same tube. If desired, it is possible to keep this final dried sample at -20 °C overnight and continue with the remaining steps the following day.
20. Add 1 mL of 0.02 M HCl to the dried extract and boil the sample for 15 min in a water bath. If the sample was frozen, leave at room temperature 5 min before boiling to avoid the fracture of the glass tube by temperature contrast.
  Note: The acid hydrolysis step is necessary to separate glycans from dolichol moieties.
21. Put the boiled samples back at room temperature and wait for them to cool down.
22. Add sequentially 3 mL of chloroform and 2 mL of methanol to each sample and mix by vortexing.
23. Centrifuge.
24. After step D23, two phases will be formed. Recover the upper phase (water phase) in a new glass tube using an eyedropper whose tip has previously been bent upwards to avoid collecting any lower phase. Remove the water phase slowly and carefully, to avoid mixing the phases.
25. Start the drying of the water phase by exposure to a constant airflow at room temperature.
26. Wash the remaining lower phase by adding 1 mL of chloroform:methanol:water (1:16:16) previously prepared and centrifuge.
27. Collect once again the upper phase using an eyedropper with the bent tip and pool it with the previous water phase while it is being dried. Wait for the pooled samples to dry completely. It is possible to pause the protocol here by keeping the dried sample at -20 °C.
Chromatography (72–82 h)
1. Resuspend the dried sample in 52 μL of Milli Q water at room temperature.
2. Check if the sample has been properly labeled with the radioactive reagents by measuring a 2 μL aliquot in a counting vial and adding 1 mL of scintillation liquid. Measure the radioactivity using a scintillation counter. Check that the aliquot has more than 200–300 CPM.
  Note: From this step, you may choose to analyze the samples by paper chromatography (protocol provided here) or by HPLC, as described in Stigliano et al. (2011).
3. Load the remaining 50 μL of the sample drop by drop on a chromatographic paper, as shown in Figure 3. Let the sample dry there before placing the paper within the chromatography chamber.
4. Run the sample by descending chromatography using 1-propanol:nitromethane:water (5:2:4). Approximately 50 mL of 1-propanol:nitromethane:water should be added to the chamber that holds the top of the paper (see Figure 3). Importantly, the run must be monitored at least once per day in order to prevent paper dryness. If required, add more solvent to the chamber. It is important to run in parallel (same paper) radioactive standards, as they will be used to identify the glycan species behind each peak (Figure 3). These standards can be obtained from a labeled chicken oviduct, as previously described by Parodi et al. (1981), or by purifying N-glycans from S. pombe strains of already known structures, as described in Fernandez et al. (1994). Once you perform this protocol and clearly identify the identity of pure peaks, you may also use those LLOs as standards. At 25 °C, a running time between 70 and 80 h would provide an optimal separation of the standard peaks, depending on the glycan species. To monitor the velocity of the chromatography, an additional standard sample can be run in parallel, on a different piece of chromatographic paper (external standard). Thus, this paper can be processed separately to obtain the position of the reference peaks at time 48 h and, based on that, decide when to stop the main running.
  
  Figure 3. Design of the chromatography paper and loading the sample. (A) Recommended layout for chromatographic paper. In this case, four running trails were drawn. At least one of these trails should be used to run in parallel an internal standard sample composed of known labeled glycan species. (B) Indication of how the chromatographic paper should be folded before hanging it in the chromatographic chamber. (C) A detail of the area where the sample should be loaded in each trail is highlighted in yellow. (D) Illustrative picture showing how to load the sample into the paper. (E) Schematic illustration of the chromatographic chamber and the chromatographic paper hung inside.
5. Remove the paper from the chromatography chamber, air dry it, and then cut each lane in 4 cm × 1 cm fractions. Place each fraction in counting vials and add 0.5 mL of scintillation liquid. It is not necessary for the liquid to cover the paper as it gets embedded.
6. Quantify the radioactive signal from each tube using a scintillation counter with a program for ¹⁴C radiation.

Data analysis

To obtain the radioactive profile of the LLO species, plot the intensity of the signal (counts per minute, CPM) versus the running length (cm) in the Excel sheet included in the following link. Figure 4A shows, as an example, representative chromatograms obtained for wildtype and Δalg8/Δalg12 S. pombe mutant strains (Gallo et al., 2022). LLOs patterns are measured in standardized conditions (see Procedure A–C of the protocol) that must be employed consistently across all experiments. Thus, as these patterns depend almost entirely on the genotype of the tested strains, the technique described here (which processes an enormous number of cells simultaneously) leads to robust and solid results, with a high level of reproducibility from replicate to replicate, and among different laboratories. Nevertheless, performing at least two independent replicates is required.

Figure 4. Representative profiles of labeled samples (A) before and (B) after deconvolution. LLO glycan patterns found in the S. pombe wildtype strain and Δalg8/Δalg12 mutant strain. In the wildtype strain, one single peak (G3M9) can be recognized, whilst in the mutant strain two not well resolved peaks (that belong to G1M7 and M7, respectively) were found. The running distance of a labeled mix of N-glycan composed by G3M9, M9, M8, M7, M6, and M5 is shown above the profiles.
Quantify the proportion of each species within a sample by a deconvolution process using a Gaussian fit applied to the obtained chromatographic profile.
Note: The data analysis strategy is to combine one or more Gaussian functions to perform a non-linear fitting of the measured profile. The included Excel sheet ‘DeconvolutionDataSheet.xlsx ’ implements the fit of up to seven independent Gaussian functions on the same profile. The procedure follows a least square fitting criterion for optimization.
Load the obtained profile in the Excel sheet as a list of Running length (cm) and Counts (CPM) paired data (cells from A12 to B61) (see Figure 5).

Figure 5. Excel Datasheet required to perform data analysis. The Excel sheet shows the number of peaks and their parameters (upper left), the data (bottom left), the plot of unprocessed data (upper right), and the processed data (bottom right). Note that a new sheet must be used for each strain.
Enable one or more Gaussian functions to fit the measured profile, setting the value ‘1’ in row 7 from columns D to J. Set the value of ‘0’ to disable.
Notes:
1. Each column from D to J corresponds to an independent Gaussian function.
2. Calculate Gaussian function values as:

Set the baseline value in cell D2 to the minimum value measured in Counts.
Note: Use a constant value as baseline in order to subtract the base signal if needed.
For each enabled function (columns D to J), set approximated values for each peak height, center, and width (rows 3–5). Use the plot on the right as a visual aid for guessing the initial parameters as close as possible to the expected result.
Sum up the baseline and all the enabled Gaussian functions for each data point (cells C12–C61).
Note: Calculate the optimization function (in cell C2) as follows:

where
Open from the Excel menu the Data/Solver.
Complete the solver parameters as follows:
Target: $C$2
To: Min
Changing cells (you must define here the range of parameters you want to use in the fitting optimization)
Method: GRG Nonlinear

Number of fitted Gaussian functions Changing cells, solver parameter
1 $D$2;$D$3:$D$5
2 $D$2;$D$3:$E$5
3 $D$2;$D$3:$F$5
4 $D$2;$D$3:$G$5
5 $D$2;$D$3:$H$5
6 $D$2;$D$3:$I$5
7 $D$2;$D$3:$J$5
Press Resolve button and wait for execution. Then, accept to keep the Solver solution.
Read the proportional area for each band from row 8, columns D–J.
Note: Calculate the area of each band from the adjusted parameters as:
Compare the profiles of duplicates and perform an average of each species in each strain.
Formulas definitions or references:
G: Value of the Gaussian function
Height: Fitting parameter indicating the maximum CPM of the peak
RL: Experimental chromatographic running length in cm
Center: Fitting parameter indicating the position of the peak maximum in cm
Width: Fitting parameter associated to the broadness of the peak in cm
𝜖: Function used by Solver to achieve the fitting
Cpm: Experimental measurement for each RL in CPM
Area: Relative quantification of the identified band

Notes

There is a limit for this deconvolution procedure to distinguish between overlapping bands.

Decreased spatial resolution may affect results.

Recipes

Yeast extract medium supplemented with adenine (YEA)
Reagent Final concentration Amount
Yeast extract 5 g/L 5 g
Glucose 30 g/L 30 g
Adenine 75 mg/L 75 mg
H₂O n/a Complete to 1 L
Total n/a 1,000 mL
Autoclave for 20 min at 120 °C
Yeast nitrogen base (YNB) without glucose
Reagent Final concentration Amount
YNB 1% 10 g
H₂O n/a Complete to 1 L
Total n/a 1,000 mL
Autoclave for 20 min at 120 °C
Radioactive solution to label a 500 μL sample
Reagent Final concentration Amount
[¹⁴C]-glucose 200 μCi 200 μL. Dry completely
Glucose (1 M) 50 mM 2.5 μL
1% YNB n/a 50 μL
Total n/a 50 μL
Chloroform:methanol:water (1:1:0.3)
Reagent Final concentration Amount
Chloroform n/a 435 mL
Methanol n/a 435 mL
H₂O n/a 130 mL
Total n/a 1,000 mL
Chloroform:methanol:water (1:16:16)
Reagent Final concentration Amount
Chloroform n/a 30 mL
Methanol n/a 485 mL
H₂O n/a 485 mL
Total n/a 1,000 mL
0.02 N HCl
Reagent Final concentration Amount
Hydrochloric acid 37% (fuming) 0.02 N 0.83 mL
H₂O n/a Complete to 500 mL*
Total n/a 500 mL
*Note: Never add water to the acid. Place some distilled water in a volumetric flask, add fuming HCl, and then add water up to 500 mL.

Acknowledgments

This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Argentina) [PICT2017-1076 to C.D’.] and the National Research Council (CONICET, Argentina) [PIP-11220150100759 to C.D]. C.D. is a Career Investigator of the National Research Council (CONICET, Argentina). A.V. and G.L.G. are CONICET fellows. This protocol derives from the original paper (Gallo et al., 2022).

Competing interests

The authors declare no competing interests.

References

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