Mapping cell surface proteolysis with plasma membrane-targeted subtiligase

Abstract

N terminomics methods combine selective isolation of protein N-terminal peptides with mass spectrometry (MS)-based proteomics for global profiling of proteolytic cleavage sites. However, traditional N terminomics workflows require cell lysis before N-terminal enrichment and provide poor coverage of N termini derived from cell surface proteins. Here, we describe application of subtiligase-TM, a plasma membrane-targeted peptide ligase, for selective biotinylation of cell surface N termini, enabling their enrichment and analysis by liquid chromatography-tandem MS (LC-MS/MS). This method provides increased coverage of and specificity for cell surface N termini and is compatible with existing quantitative LC-MS/MS workflows.

1. Introduction

Proteolysis is an important post-translational modification that regulates the function, subcellular localization, and lifetime of most proteins [1, 2]. At the cell surface, proteolytic protein modification is widespread, regulating cell-cell communication and the cellular response to environmental cues [3]. Mass spectrometry (MS)-based proteomics methods have emerged as powerful tools for protease substrate identification with single amino acid resolution, enabling global profiling of the proteolytic response to biological stimuli [4–11]. However, these techniques, collectively known as ‘N terminomics’ methods, provide limited information about cleavage events in cell surface proteins, which often evade detection based on the lower abundance of cell surface proteins compared to intracellular proteins [12, 13]. In this chapter, we provide a detailed protocol for application of subtiligase-TM, a cell surface-tethered variant of the designed peptide ligase subtiligase, for specific biotinylation of protein N termini that are displayed on the extracellular surface (Figure 1) [14]. Following biotinylation, the modified, cell surface-derived N-terminal peptides can be sequenced and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Figure 1. Subtiligase-TM workflow.

A stable cell line expressing subtiligase-TM is treated with a biotinylated subtiligase substrate (TEV Ester 6) to effect biotinylation of cell surface N termini. After labeling, the cells are lysed, biotinylated proteins are enriched on neutravidin resin, and a trypsin digestion is performed to remove internal peptides. N-terminal peptides are then selectively eluted by cleavage of a TEV protease site found in the subtiligase substrate and are analyzed by LC-MS/MS.

Proteolytic cleavage events have the universal feature that they generate neo-N and neo-C termini at the site of cleavage. N terminomics methods take advantage of the unique structure and/or reactivity of the protein N terminus compared to lysine ε-amines to isolate N-terminal peptides for sequencing with single amino acid resolution using LC-MS/MS and are therefore a valuable tool for the study of proteolytic signaling. One class of N terminomics techniques relies of positive enrichment of N-terminal peptides following selective N-terminal biotinylation [10, 11], while another class uses strategies to deplete internal tryptic peptides from the sample [4–9]. Both strategies typically rely on isolation of N-terminal peptides after cell lysis, precluding selective isolation of cell surface proteins. While N terminomics methods can be combined with subcellular fractionation approaches, these techniques often suffer from low specificity and lead to sample losses.

Subtiligase-TM N terminomics is a recently developed method that takes advantage of the genetic targetability of enzymes for selective biotinylation of cell surface N termini [14]. Subtiligase is a designed peptide ligase that has been used extensively in the context of cell lysate for N terminomics studies and whose specificity has been engineered to provide high coverage of the cellular N terminome [15, 16]. Subtiligase-TM was generated by fusing subtiligase to a transmembrane domain derived from the platelet-derived growth factor receptor β chain (PDGFRβ). By expressing subtiligase-TM in cells, subtiligase activity can be restricted to the cell surface for efficient and selective biotinylation of N termini before lysis, while membranes and spatial relationships remain intact. Labeling is initiated by addition of a cell-impermeable biotinylated peptide ester substrate, TEV Ester 6 (Figure 2), which harbors a TEV protease cleavage site for selective elution and an ⍺-aminobutyric acid (Abu) mass tag for positive identification of subtiligase modified peptides. Following labeling, the biotinylated N termini can be isolated and analyzed by LC-MS/MS. After construction of a stable cell line harboring subtiligase-TM, the cell surface N terminomics workflow can be completed within two days. A typical experiment leads to identification of 300–500 cell surface-derived N-terminal peptides from one 500 mm² dish of cells.

Figure 2. Structure of TEV Ester 6 (TE6).

TE6 is a peptide ester with the sequence biotin-EEENLYFQ-⍺-aminobutyric acid-glycolate ester-R. The peptide is typically synthesized as a C-terminal amide.

2. Materials

All buffers should be made with ultrapure water.

2.1. Cell culture and cell line construction

1. HEK293T cell line (ATCC CRL-11268)

2. 5% CO2 37°C tissue culture incubator

3. 6-well sterile tissue culture plates

4. Complete DMEM: Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 100 units/mL penicillin G, 100 µg/mL streptomycin, and 10% fetal bovine serum

5. Serum-free DMEM

6. FuGENE HD transfection reagent (Promega) (see Note 1)

7. pCMV-ΔR8.91 (Creative Biogene) and pMD2.G (Addgene #12259) second-generation lentiviral packaging plasmids (see Note 2)

8. pLX302-subtiligase TM (available upon request from Dr. James A. Wells, University of California, San Francisco)

9. 0.45 µm PVDF syringe filter

10. Polybrene (4 mg/mL)

11. Puromycin (2 mg/mL)

2.2. Subtiligase cell labeling

1. Phosphate-buffered saline (PBS)

2. Labeling buffer: 100 mM tricine, pH 8.0, 150 mM NaCl

3. Subtiligase substrate TEV Ester 6 (TE6) [17]: A synthetic peptide with the sequence biotin-EEENLYFQ-⍺-aminobutyric acid-glycolate ester-R-amide (Figure 2; see Note 3)

4. Refrigerated microcentrifuge

2.3. Fluorescence imaging

1. 35 mm poly-d-lysine-coated glass bottom dishes

2. PBS containing 3% bovine serum albumin (BSA)

3. 1% paraformaldehyde

4. Fluorescence microscope

5. AlexaFluor 647-conjugated anti-DYKDDDK tag antibody (BioLegend #637315)

6. AlexaFluor 488-conjugated streptavidin (BioLegend #405235)

2.4. Cell harvest and cell lysis

1. 500 cm² sterile tissue culture dishes

2. Versene: PBS containing 0.53 mM EDTA (see Note 4)

3. RIPA lysis buffer: 50 mM Tris-HCl, pH 7.4, 1 M NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate

4. Halt Protease Inhibitor Cocktail (Thermo Scientific)

5. Probe ultrasonicator equipped with a microtip

2.5. N-terminal peptide enrichment

1. High-capacity neutravidin agarose resin (Thermo Fisher cat. no. 29202)

2. 1 mL snap-cap spin columns suitable for attachment to a vacuum manifold (see Note 5)

3. PBS containing 1 M NaCl

4. 100 mM ammonium bicarbonate (see Note 6)

5. 100 mM ammonium bicarbonate containing 2 M urea (see Notes 6, 7)

6. 1 M tris(2-carboxyethyl)phosphine (TCEP) (see Note 8)

7. 500 mM iodoacetamide (see Note 9)

8. Sequencing-grade modified trypsin

9. 4 M guanidine hydrochloride

10. 1 M dithiothreitol (DTT)

11. Tobacco etch virus (TEV) protease (see Note 10)

12. 5% trifluoracetic acid (TFA)

2.6. Peptide desalting

1. C18 desalting spin tips or columns (see Note 11)

2. Conditioning buffer: 80% acetonitrile, 20% water, 0.1% TFA

3. Wash buffer: 0.1% TFA

4. Elution buffer: 0.1% formic acid, 50% acetonitrile

5. Vacuum concentrator

2.7. LC-MS/MS analysis

1. MS buffer A: 0.1% formic acid in LC-MS grade water

2. MS buffer B: 0.1% formic acid, 80% LC-MS grade acetonitrile, 20% LC-MS grade water

3. Acclaim PepMap RSLC column (75 µm x 15 cm, 2 µm particle size, 100 Å pore size) (see Note 12)

4. Thermo Dionex UltiMate 3000 RSLCnano LC system coupled to a Thermo QExactive Plus hybrid quadrupole-Orbitrap mass spectrometer (see Note 13)

3. Methods

3.1. Lentivirus production

1. The day before transfection, seed one well of a 6-well plate with 6–9 × 10⁵ HEK293T cells in 2.6 mL complete DMEM. Incubate at 37ºC, 5% CO₂ for 24 h.

2. Immediately before transfection, mix 300 µL serum-free DMEM with 7.5 µL FuGENE HD transfection reagent. Incubate for 5 min at room temperature (see Note 1).

3. In the cap of the tube containing the FuGENE HD mixture, mix 1.35 µg pCMV-ΔR8.91, 0.165 µg of pMD2.G and 1.5 µg of pLX302-subtiligase-TM. Close the lid and mix. Incubate for 30 min at room temperature (see Note 2).

4. Transfer the plasmid mixture into the well containing HEK293T cells. Incubate at 37ºC, 5% CO₂ for 72 h to allow viral production to proceed.

3.2. Lentivirus infection of HEK293T cells

1. The day before transfection, seed one well of a 6-well plate with 6–9 × 10⁵ HEK293T cells in 2.6 mL complete DMEM. Incubate at 37ºC, 5% CO₂ for 24 h.

2. Remove virus-containing supernatant from HEK293T cells and filter through a 0.45 µm PVDF filter into a 50 mL conical tube.

3. Aspirate media from HEK293T cells to be infected. Add 2 mL of fresh complete DMEM, followed by 6 µL of 4 mg/mL polybrene. Swirl gently to mix.

4. Add 1 mL of filtered virus. Incubate at 37ºC, 5% CO₂ for 24 h.

5. Aspirate media. Replace with 3 mL complete DMEM. Incubate at 37ºC, 5% CO₂ for 48 h.

6. Add puromycin to a final concentration of 2 µg/mL to select for lentivirally transduced cells (see Note 12).

3.3. Fluorescence imaging to validate subtiligase-TM expression and activity

1. Seed a 35 mm poly-d-lysine coated glass bottom dish with 6–9 × 10⁵ subtiligase-TM-transduced HEK293T cells in 3 mL complete DMEM one day before the imaging experiment is to be performed.

2. On the day of the experiment, aspirate media from cells. Wash cells three times with PBS, being careful not to disturb the cells.

3. Add 1 mL of 2.5 mM TE6 dissolved in labeling buffer (see Note 3). Incubate at room temperature for 20 min.

4. Aspirate labeling solution. Wash cells three times with PBS, being careful not to disturb the cells.

5. Fix cells by adding 1% paraformaldehyde in PBS. Incubate cells at room temperature for 10 minutes. Optionally, cells may also be permeabilized by adding 0.5% Triton X-100 to the fixing solution.

6. Block cells by washing with PBS containing 3% BSA.

7. Dilute AlexaFluor 647-conjugated anti-DYKDDDK tag antibody to 0.5 µg/mL and AlexaFluor 488-conjugated streptavidin to 0.5 µg/mL in 2 mL PBS containing 3% BSA. Add staining solution to cells and incubate in the dark for 30 min at room temperature.

8. Aspirate staining solution. Wash cells three times with PBS containing 3% BSA.

9. Add PBS to cells and image with a fluorescence microscope. Typical fluorescence images are shown in Figure 3.

Figure 3. Fluorescence images of HEK293T cells stably expressing subtiligase-TM.

Subtiligase-TM (SL-TM) expression is detected with an anti-FLAG antibody conjugated to Alexa Fluor 647 and biotinylation activity is detected with streptavidin conjugated to Alexa Fluor 488.

3.4. Cell harvest

Example with one 500 cm² dish of HEK293T-Subtiligase-TM cells grown to ~80% confluency.

1. Aspirate media from cells.

2. Wash cells twice with 25 mL PBS, taking care not to detach the cells from the plate.

3. Add 25 mL versene to culture dish and swirl to completely cover the cells. Incubate at 37ºC for 5–10 minutes to allow cells to detach. Tap sides of the dish gently every few minutes to further facilitate detachment (see Note 4).

4. After cells have detached, resuspend them in the versene and transfer to a 50 mL conical tube.

5. Centrifuge for 5 min at 300 × g. Aspirate the supernatant.

6. Wash cells in 50 mL ice-cold PBS. Centrifuge at 300 × g and aspirate the supernatant.

7. Resuspend the cells in 1 mL ice-cold PBS and transfer to a 1.5 mL low-retention microcentrifuge tube. Centrifuge at 300 × g and aspirate the supernatant.

3.5. Subtiligase-TM labeling

1. Wash cells twice in 1 mL ice-cold subtiligase-TM labeling buffer. Centrifuge at 300 × g and aspirate the supernatant.

2. Resuspend cells in 1 mL ice-cold PBS. Transfer 50 µL of cell suspension to a separate microcentrifuge tube as a pre-labeling sample for Western blot analysis. Centrifuge both tubes at 300 × g for 5 min. Aspirate the supernatant. Freeze the cells from the 50 µL sample at −80ºC and proceed with labeling using the remaining cells.

3. Add 1 mL of 2.5 mM TE6 dissolved in labeling buffer (see Note 13).

4. Place tube on a rotating mixer at 4ºC for 1 h.

5. After 1 h, remove 50 µL of the cell suspension from the tube to save as a post-labeling sample for Western blot analysis. Centrifuge at 300 × g, aspirate the supernatant, and freeze the cells at −80ºC.

6. Centrifuge the remaining labeled cells at 300 × g for 5 min at 4ºC and aspirate the supernatant.

7. Resuspend the cells in 1 mL ice-cold PBS. Centrifuge for 5 min at 4ºC and aspirate the supernatant. Repeat two additional times to wash away excess TE6.

3.6. Cell lysis

1. Prepare 1 mL RIPA lysis buffer per sample. Immediately before use, add 10 µL 0.5 M EDTA and 10 µL Halt Protease Inhibitor Cocktail.

2. Resuspend the cells in 1 mL RIPA lysis buffer.

3. Sonicate cell suspension using a microtip probe for 10 cycles of 5 s on / 5 s off at 20% amplitude.

4. Pellet the cell debris by centrifuging at 13,000 × g for 10 min at 4ºC in a microcentrifuge.

5. Transfer supernatant to a new 1.5 mL tube.

3.7. Biotinylated protein enrichment

1. Mix High-Capacity NeutrAvidin Agarose resin to generate a uniform 50% slurry, then transfer 500 µL of the slurry into a 1.5 mL low-retention tube for each sample.

2. Centrifuge the resin at 500 × g at room temperature. Discard the supernatant.

3. Resuspend the resin in 1 mL RIPA buffer. Centrifuge the resin for 2 min at 500 × g at room temperature and discard the supernatant. Repeat two additional times.

4. Add the cell lysate to the resin and incubate for 1 h at room temperature on a rotator or mixer.

5. Centrifuge the resin for 2 min at 500 x g at room temperature. Save the supernatant for Western blot analysis.

6. Resuspend the resin in 500 µL of RIPA buffer. Transfer to a 1 mL snap-cap column and attach to vacuum manifold.

7. Wash the resin 10 times with 800 µL RIPA buffer.

8. Wash the resin 10 times with 800 µL PBS containing 1 M NaCl.

9. Wash the resin 10 times with 800 µL 100 mM ammonium bicarbonate.

10. Wash the resin 10 times with 800 µL 100 mM ammonium bicarbonate with 2 M urea.

3.8. Reduction, alkylation, and trypsin digestion

1. Remove the column from the vacuum manifold and cap the bottom. Resuspend resin in 1 mL ammonium bicarbonate with 2 M urea and transfer to a 1.5 mL low-retention tube.

2. Add 1 M TCEP to a final concentration of 5 mM. Incubate the sample on a rotating mixer for 30 min.

3. Add 500 mM iodoacetamide to a final concentration of 10 mM and incubate on a rotating mixer for 1 h at room temperature in the dark (see Notes 9, 14).

4. Centrifuge the resin at 500 x g at room temperature for 2 min. Discard the supernatant.

5. Resuspend the resin in 1 mL ammonium bicarbonate with 2 M urea. Centrifuge the resin at 500 x g at room temperature for 2 min. Discard the supernatant. Repeat two additional times.

6. Resuspend the resin in 1 mL 100 mM ammonium bicarbonate with 2 M urea. Add 20 µg sequencing-grade modified trypsin. Incubate at room temperature for 12–16 h on a rotating mixer.

7. Centrifuge resin for 2 min at 500 x g at room temperature. Discard the supernatant.

8. Resuspend the resin in 800 µL of 100 mM ammonium bicarbonate with 2 M urea and transfer to a new snap-cap spin column. Place the column on vacuum manifold.

9. Wash the resin 10 times with 800 µL 100 mM ammonium bicarbonate with 2 M urea.

10. Wash the resin 10 times with 800 µL 100 mM ammonium bicarbonate.

11. Wash the resin 10 times with 800 µL 4 M guanidinium hydrochloride.

12. Wash the resin 10 times with 800 µL 100 mM ammonium bicarbonate.

3.9. TEV protease elution of N-terminal peptides

1. Remove the column from the vacuum manifold and cap the bottom. Resuspend the resin in 500 µL of 100 mM ammonium bicarbonate and transfer to a 1.5 mL low-retention tube.

2. Add 2 µL of 1 M DTT (see Note 15).

3. Add 10 µg TEV protease. Incubate on a rotating mixer for 2–6 h or up to overnight.

4. Centrifuge for 2 min at 500 x g at room temperature. Place a clean snap-cap column into a 1.5 mL low-retention tube. Resuspend the resin in the supernatant and transfer it into the column.

5. Centrifuge for 2 min at 500 x g at room temperature. Save the flowthrough, which contains the eluted N-terminal peptides.

6. Wash the resin with 250 µL of 100 mM ammonium bicarbonate. Combine the wash with the supernatant from step 5.

7. Dry the combined eluate in a vacuum concentrator.

8. Resuspend the dried sample in 50 µL of 5% TFA to precipitate the TEV protease. Incubate on ice for 10 min.

9. Centrifuge for 10 min at 20,000 x g at 4ºC to pellet the precipitated protease.

3.10. Sample desalting

Example with Pierce C18 spin tips (see Note 11).

1. Condition the spin tip by adding 20 µL conditioning buffer. Spin for 1 min at 1000 × g.

2. Equilibrate the tip by adding 20 µL wash buffer. Spin for 1 min at 1000 × g.

3. Load the sample by adding 20 µL of the supernatant from 3.9, step 9. Spin for 1 min at 1000 × g. Perform additional spins to load the entire sample. Visually inspect the tip to ensure that the sample has flowed through; if sample remains in the tip, spin at 1000 × g until it flows through completely.

4. Wash the tip with 20 µL wash buffer. Spin for 1 min at 1000 × g. Repeat one additional time.

5. Place the tip in a clean tube. Elute the sample by adding 20 µL elution buffer. Spin for 1 min at 1000 × g. Repeat one additional time.

6. Evaporate the sample to near-dryness in a vacuum concentrator.

3.11. LC-MS/MS and data analysis

Example using an Acclaim PepMap RSLC column (75 µm x 15 cm, 2 µm particle size, 100 Å pore size) using Thermo Dionex UltiMate 3000 RSLCnano LC system coupled to a Thermo QExactive Plus hybrid quadrupole-Orbitrap mass spectrometer.

1. Dissolve the peptides in 10 µL of 0.1% formic acid containing 2% acetonitrile for MS analysis (see Note 16).

2. Load 5 µL of sample onto the column over 15 min at 0.5 µL/min MS buffer A. Elute peptide at 0.3 µL/min using a linear gradient from MS buffer A to 40% MS buffer B over 125 min. Perform data-dependent acquisition using a scanning mass range from 300–1,500 m/z using Thermo Xcalibur software.

3. Search the data again the human SwissProt database using Thermo Scientific Proteome Discoverer with SEQUEST HT (see Note 17). Set the enzyme to Trypsin (Semi) to enable identification of proteolytic neo-N termini generated by endogenous proteases. Set carbamidomethylation of cysteine (+57.021 Da) as a static modification. Set the following dynamic modifications: aminobutyric acid (Abu, +85.053 Da) at peptide N termini; acetylation (+42.011 Da), methionine loss (−131.040 Da) and methionine loss + acetylation (−89.030 Da) at protein N termini; and methionine oxidation (+15.995 Da).