May 14, 2025

Bioorthogonal Non-Canonical Amino Acid Tagging (BONCAT) to Detect Newly Synthesized Proteins in Cells and their Secretome

PLOS One
Peer-reviewed method
DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6yw5zvqe/v1
  • Elizabeth P. Anim1,
  • Justin Mezzanotte1,
  • Siwei Chu1,
  • Ursula Stochaj1,2
  • 1Department of Physiology, McGill University, Montreal, Quebec, Canada;
  • 2Quantitative Life Sciences Program, McGill University
Ursula Stochaj
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6yw5zvqe/v1
External link: https://doi.org/10.1371/journal.pone.0329857
Protocol Citation: Elizabeth P. Anim, Justin Mezzanotte, Siwei Chu, Ursula Stochaj 2025. Bioorthogonal Non-Canonical Amino Acid Tagging (BONCAT) to Detect Newly Synthesized Proteins in Cells and their Secretome. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6yw5zvqe/v1
Manuscript citation:
Anim EP, Mezzanotte J, Chu S, Stochaj U (2025) Bioorthogonal Non-Canonical Amino Acid Tagging (BONCAT) to detect newly synthesized proteins in cells and their secretome. PLOS One 20(8). doi: 10.1371/journal.pone.0329857
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it is working.
Created: April 30, 2025
Last Modified: May 14, 2025
Protocol  Integer ID: 218224
Keywords: de novo protein synthesis, non-canonical amino acid, L-azidohomoalanine, biotinylation, affinity purification, secretome analysis, Bioorthogonal Non-Canonical Amino Acid Tagging (BONCAT) , bioorthogonal noncanonical amino acid tagging, changes in de novo protein synthesis, de novo protein synthesis, synthesized protein, produced protein, synthesized proteins in cell, secretome change, secretome cell, protein, de novo in mammalian cell, secretome, composition of the protein, biotinylated polypeptide, translated polypeptide chain, biotin affinity tag, azidohomoalanine, de novo, translated polypeptide, subsequent affinity purification, proteome, secretion, compatible with the subsequent affinity purification, polypeptide chain, methionine analog
Funders Acknowledgements:
NSERC
Grant ID:
Abstract
Cells respond to physiological or pathological stimuli by altering the composition of the proteins they produce. This adaptation includes changes to newly translated polypeptides that are destined for intracellular compartments or secretion. The secretome is relevant to cell physiology, as it promotes a noutocrine, paracrine, and endocrine signaling. These events control cell death, tissue repair and other regenerative processes. Uncovering the changes in de novo protein synthesis under different growth conditions requires reliable methods to identify and quantify newly synthesized proteins. Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) can generate this information with high spatiotemporal resolution.
We developed a BONCAT-based protocol to characterize proteins synthesized de novo in mammalian cells. The current protocol employs L-azidohomoalanine as an L-methionine analog, which is incorporated into newly translated polypeptide chains. After the incubation period, cells and the growth medium, which contains the secretome, are processed separately. Specifically, proteins are alkylated, and L-azidohomoalanine is modified with a biotin affinity tag. Proteins are collected using a rapid precipitation method, which is compatible with the subsequent affinity purification of biotinylated polypeptides. The affinity-purified material can be used for diverse downstream applications, such as Western blotting.
Our modified BONCAT protocol was developed to study newly produced proteins in growing cells and their secretome. This method will be useful to examine the proteome and secretome changes that are linked to the altered performance of cells, tissues, and organs during aging, disease, or other challenging conditions.
Guidelines
Follow general laboratory safety practices.

Thumbnail image:



Additional Notes:

  1. Store cell extracts and medium/secretome fraction at -70ºC if not used immediately. Avoid freeze-thawing the samples to prevent protein degradation.
  2. Prepare AHA stock solution fresh in sterile water.
Materials
Biological materials:

  • HeLa cells (ATCC, Catalog #: CCL-2)

Reagents:

  1. DMEM, high glucose, no glutamine, no methionine, no cystineThermo FisherCatalog #21013024
  2. 0.05% Trypsin/ 0.53mM EDTA, 1X 500mLWisent BioproductsCatalog #325-542 CL
  3. HEPES 1M Free acidWisent BioproductsCatalog #330-050-EL
  4. 1% Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140-122
  5. Sodium Pyruvate 100 mMWisent BioproductsCatalog #600-110-EL
  6. L-GlutamineTCI ChemicalsCatalog #G0063
  7. Bovine Calf Serum (BCS)Fisher ScientificCatalog #SH3734IR254
  8. L-(-)-Cystine DihydrochlorideTCI ChemicalsCatalog #C0520
  9. L-Azidohomoalanine HCl saltBroadPharmCatalog #BP-23383
  10. DBCO-PEG4-biotinBroadPharmCatalog #BP-22295
  11. Iodoacetic acid (ICN Biomedicals, Catalog #: 100351)
  12. UltraPure™ Sodium Dodecyl Sulfate (SDS)Thermo Fisher ScientificCatalog #15525017
  13. Methanol, MetOH (ThermoFisher Scientific, Catalog #: BP1105-4)
  14. Chloroform, CHCl3, 99.8% (BDH, ACS)
  15. Streptavidin MagBeadsGenscriptCatalog #L00936
  16. Streptavidin - HRPThermo Fisher ScientificCatalog #434323
  17. Nitrocellulose Membrane 0.45 umBio-Rad LaboratoriesCatalog #1620115
  18. SuperSignal™ West Pico PLUS Chemiluminescent SubstrateThermo Fisher ScientificCatalog #34580 or SuperSignal™ West Atto Ultimate Sensitivity SubstrateThermo Fisher ScientificCatalog #A38554

Solutions and recipes:

All solutions are prepared in distilled water

  • 1M HEPES
  • PBS, per L:
AB
NaCl8 g
KCl0.2 g
Na2HPO41.44 g
KH2PO40.24 g
Adjust to pH 7.4, autoclave

  • 20% (w/v) SDS in water
  • 2% (w/v) SDS in PBS
  • L-Azidohomoalanine HCl salt, 50 mM stock solution in sterile water
  • Iodoacetic acid (IAA), 200 mM in water; prepare fresh solution for each experiments
  • N-ethylmaleimide (NEM), 200 mM in water; prepare fresh solution for each experiments; alternative for IAA
  • 1 mM DBCO-PEG4-biotin in water
  • Sample buffer for SDS-PAGE, pH 8.0; 2-fold concentrated (2X):
To prepare 10 ml of 2X sample buffer, mix the following

AB
Tris (hydroxymethyl) amino methane, pH 8.01.6 mL
50% glycerol4.6 mL
SDS0.4 g
Dithiothreitol308 mg
0.4% Bromophenol Blue0.1 mL
β-glycerophosphate86 mg
Protease inhibitor cocktail, 2X, (Roche, EDTA-free)
NaF5mM
NaN32mM
Keep aliquots frozen at -20ºC.

  • Running buffer for SDS-PAGE:

5X stock solution, for 1 L dissolve

AB
Tris (hydroxymethyl) amino methane15 g
Glycine72 g
SDS5 g
Adjust pH to 8.3 with concentrated HCl
Keep stock solution at 4 ºC

  • Blotting buffer:

For 700 mL, dissolve in distilled water

AB
Tris (hydroxymethyl) amino methane2.13 g
Glycine10.12 g

  • Tris-buffered saline containing 10mM Tris (hydroxymethyl) amino methane, 150 mM NaCl, 0.1 % Tween-20, adjust to pH 7.6 with HCl.

Laboratory equipment:

  • BioLite™ Cell Culture Treated Flasks, 75 cm² (ThermoFisher Scientific, Catalog #: 130190)
Equipment
BioLite™ Cell Culture Treated Flasks, 75 cm2
NAME
Flask
TYPE
Thermo Scientific™
BRAND
130190
SKU
https://www.thermofisher.com/order/catalog/product/130190?SID=srch-srp-130190
LINK

  • Tissue culture dish, 35 x 10 mm (Sarstedt, Catalog #: 83.3900)
Equipment
Tissue culture dish, (ØxH): 35 x 10 mm, surface: Standard
NAME
Culture plates
TYPE
Sarstedt
BRAND
83.3900
SKU
https://www.sarstedt.com/en/products/laboratory/cell-tissue-culture/cultivation/product/83.3900/
LINK

  • Hermle, Labnet tabletop centrifuge
  • Microfuge (Eppendorf 5424/5424R, Catalog #: EP5404000537)
  • Bio-Rad Mini-PROTEAN Cell Gel System
  • Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell
  • Sonicator bath; ultrasonic frequency 40 kHz (Codyson, Model PS-10A)
  • ChemiDoc™ MP Imaging System (Bio-Rad, Catalog #: 17001401)





Safety warnings
Safety Warnings

  • Several of the compounds used in this protocol are hazardous to human health. The list below is not complete. PubChem (https://pubchem.ncbi.nlm.nih.gov) provides additional information. Safety Data Sheets should be consulted prior to the application of the protocol.
  • Iodoacetic acid is corrosive and acute toxic. N-ethylmaleimide (NEM) is corrosive, acute toxic, and an irritant. Chloroform is a health hazard with acute toxicity. Sodium dodecyl sulfate is flammable, corrosive, and an irritant.
Before start
Determine the possible toxicity of L-azidohomoalanine for the experimental model system to be used. Adjust the final concentration of AHA, if necessary.
Part I. Cell culture conditions for the incorporation of AHA and control experiments
BONCAT medium

Thaw sterile aliquots of BCS and Pen/Strep.

Warm L-glutamine solution in water bath (30ºC - 32ºC); vortex repeatedly until solution is clear.

Supplement 500 mL of DMEM with:

AB
L-glutamine stock (final concentration: 4 mM)5 mL
L-cystine (final concentration: 0.201 mM)31.5 mg
1 M HEPES buffer 5 mL

Adjust pH to 7.1, add HCl dropwise.

Filter the prepared medium into a sterile bottle using a vacuum-driven filter system.

Add BCS (8% final concentration), 5 mL Pen/Strep, and 5 mL of 100 millimolar (mM) sodium pyruvate aseptically.

Mix content thoroughly and store at 4 °C until use.

Part I/II: Cells are grown in medium supplemented with AHA. AHA is taken up by cells and incorporated into newly synthesized proteins.

Part II. Biotinylation of AHA-modified polypeptides
3h 5m
A. Treatment of medium/secretome fraction

Grow cells in 3 cm dishes, treat cells according to protocol; incubate cells in BONCAT medium containing AHA or supplemented with L-methionine (control samples).

After AHA incorporation (or L-methionine controls) collect medium (2 mL , contains secretome) into 15 mL tube.

Spin at 720 x g, Room temperature, 00:05:00 , to remove debris and floating cells.

5m
Transfer supernatant to fresh 15 mL tube. Do not touch the sediment.

Add SDS (20% stock solution) to 2% final concentration.

Add freshly prepared IAA (or NEM) to 50 millimolar (mM) final concentration.

Rotate 00:30:00 ,Room temperature , protect from light.

30m
Add DBCO-PEG4-biotin to 1 micromolar (µM) final concentration.

Note
The “medium” fraction is approximately 10 times the volume of the “cell” fraction.

Rotate 01:00:00 , Room temperature , protect from light.

1h
Store medium at -70 °C or use immediately for MetOH/CHCl3 extraction and affinity-purification with Streptavidin-MagBeads.

B. Treatment of cells

After removal of medium, wash dish once with 2 mL PBS, Room temperature .

Add 100 µL PBS/2% SDS. Scrape material into 1.5 mL tube; Room temperature .

Rinse dish with 100 µL PBS/2% SDS. Add to test tube; Room temperature .

Note
The solution is highly viscous.

Add freshly prepared IAA (or NEM) to 50 millimolar (mM) final concentration.

To shear DNA, sonicate the sample twice for 5 min at RT; protect sample from light.

  • To shear DNA, sonicate the sample for 00:05:00 at Room temperature ; protect sample from light. (1/2)
  • To shear DNA, sonicate the sample for 00:05:00 at Room temperature ; protect sample from light. (2/2)

10m
Rotate 00:20:00 at Room temperature , protect from light. (Total incubation time with IAA is 30 min.)

20m
Add DBCO-PEG4-biotin to 10 micromolar (µM) final concentration.

Rotate 01:00:00 , Room temperature , protect from light.

1h
Store cell extract at -70 °C or use immediately for MetOH/CHCl3 extraction and affinity-purification with Streptavidin MagBeads.

Part II. Click chemistry modifies AHA incorporated into different polypeptides with a biotin moiety.

Part III. Precipitation of proteins present in growth medium/secretome and cell fractions
50m 10s

Note
The protocol is described for 100 µL extracts. It can be scaled up for larger volumes. All steps are performed at Room temperature .

Mix 100 µL of extracts described in Part II (Step 8.10 of Part II-A or Step 9.9 of Part II-B) with 400 µL MetOH.

Add 100 µL CHCl3, vortex.

Add 300 µL distilled water, vortex.

Spin 14000 x g, 00:05:00 .

5m
Carefully remove the clear upper phase. Do not disturb the white precipitate at interphase.

Add 400 µL MetOH, vortex.

Spin 20000 x g, 00:05:00 . Carefully remove the clear upper phase. Do not disturb precipitate.

5m
Spin 20000 x g, 00:00:10 . Remove residual liquid.

10s
Dry sediment at Room temperature for ~00:30:00 .

30m
Resuspend precipitate in 500 µL PBS/1% SDS.

Sonicate two times, 5 min for each step.

Sonicate, 00:05:00 for each step. (1/2)

5m
Sonicate, 00:05:00 for each step. (2/2)

5m
Proceed to affinity purification or store at -70 °C .

Part III. A complex mixture of proteins includes de novo synthesized and biotinylated polypeptides. Proteins are extracted and precipitated. The precipitate is resuspended in an aqueous buffer for subsequent purification.

Part IV. Affinity purification of biotinylated proteins
1h 20m 15s
Dilute material precipitated with MetOH/CHCl3 1:2 with PBS. The sample is now in PBS/0.5% SDS.

Spin 15871 x g, Room temperature, 00:05:00 .

5m
Use supernatant for incubation with Streptavidin MagBeads.

Wash 75 µL of Streptavidin MagBeads three times with 750 µL PBS/0.5% SDS.

Add beads to supernatant obtained in Step 24.

Incubate 01:00:00 at Room temperature , rotate sample.

1h
Spin 00:00:05 in microfuge.

5s
Insert into magnet. Aspirate liquid with vacuum.

Wash beads three times with 750 µL PBS/0.1% SDS.

Add 80 µL 2X sample buffer, vortex, spin 00:00:05 in microfuge,

5s
Incubate 00:15:00 at 95 °C .

15m
Spin 00:00:05 in microfuge.

5s
Insert into magnet and transfer liquid to fresh tube.

Separate samples by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE).

Part IV/V. Affinity purification enriches for biotinylated proteins. These proteins are characterized by different downstream applications.

Part V. Blotting and analysis of de novo synthesized proteins
11h 40m
Run samples on SDS-PA gels.

Blot proteins onto nitrocellulose filter for 01:20:00 at 76 V, constant voltage.

1h 20m
Rinse filter two times for 5 min in TBST.

Rinse filter for 00:05:00 in TBST. (1/2)

5m
Rinse filter for 00:05:00 in TBST. (2/2)

5m
Block filter in TBST/5% non-fat milk powder, 01:00:00 at Room temperature .

1h
Rinse filter two times for 5 min in TBST.

Rinse filter for 00:05:00 in TBST. (1/2)

5m
Rinse filter for 00:05:00 in TBST. (2/2)

5m
Agitate filter Overnight in cold room with Streptavidin-HRP, diluted 1:5,000 in TBST/1% non-fat milk powder.

8h
Wash filter for 01:00:00 in TBST at Room temperature ; change buffer every 15 min.

1h
Incubate filter with ECL substrate.

Collect ECL signals with ChemiDoc™ MP Imaging system.

Acknowledgements
Funding: Fonds de recherche du Québec, Nature et technologies (FRQNT), Natural Sciences and Engineering Research Council of Canada (NSERC).