TIE-UP-SIN：一种提高蛋白质相互作用鉴定效率的方法

Maximilian Schedlowski; Stephan Michalik; Tilly Hoffmüller; Marco Harms; Leif Steil; Kristin Surmann; Christian Hentschker; Manuela Gesell Salazar; Uwe Völker; Alexander Reder

doi:10.21769/BioProtoc.5663

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Peer-reviewed

TIE-UP-SIN: A Method for Enhanced Identification of Protein–Protein Interactions

TIE-UP-SIN：一种提高蛋白质相互作用鉴定效率的方法

MS Maximilian Schedlowski

CH Christian Hentschker

MS Manuela Gesell Salazar

UV Uwe Völker

AR Alexander Reder email

发布: 2026年04月20日第16卷第8期 DOI: 10.21769/BioProtoc.5663 浏览次数: 385

评审: Alessandro DidonnaKasturika ShankarAnonymous reviewer(s)

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Abstract

Protein–protein interactions (PPIs) govern nearly all aspects of cellular physiology, yet identifying these interactions under native conditions remains challenging. Here, we present TIE-UP-SIN (targeted interactome experiment for unknown proteins by stable isotope normalization), a robust method for in vivo identification and quantification of PPIs in bacterial systems. The protocol combines metabolic labeling with ¹⁵N isotopes, reversible formaldehyde crosslinking, affinity purification, and quantitative mass spectrometry. TIE-UP-SIN preserves transient or weak interactions during purification and quantifies interaction partners using internal light/heavy peptide ratios, reducing experimental variability. The method employs a triple-sample design to distinguish specific from nonspecific interactors and can be adapted to various bacterial species and affinity tags. Data analysis is streamlined through a user-friendly web application (https://shiny-fungene.biologie.uni-greifswald.de/TIE_UP_SIN_app) that automates statistical analysis, normalization, and visualization, requiring no programming expertise. The entire workflow from cell culture to mass spectrometry data acquisition takes approximately 4–5 days, with data analysis completed in 1–2 days using the web application.

Key features

• Captures transient protein interactions in vivo through reversible formaldehyde crosslinking under native expression conditions.

• Internal ¹⁵N metabolic labeling enables robust quantification and reduces experimental variability across biological replicates.

• Triple-sample design (WT/WT, bait/WT, bait/bait) distinguishes specific from nonspecific interactors with high confidence.

• Applicable to diverse bacterial systems with simple adaptation to any affinity-tagged bait protein.

Keywords: Protein–protein interactions (蛋白质相互作用)

Heavy nitrogen metabolic labeling (重氮代谢标记)

Formaldehyde crosslinking (甲醛交联)

In vivo crosslinking (体内交联)

Mass spectrometry (质谱分析)

Affinity purification–mass spectrometry (亲和纯化-质谱)

Quantitative proteomics (定量蛋白质组学)

Background

Identifying protein–protein interactions (PPIs) under physiologically relevant conditions is essential for understanding cellular function. Traditional methods such as affinity purification–mass spectrometry (AP–MS) primarily capture stable interactions, while transient or weak binding partners may be lost during purification. Proximity labeling techniques like BioID and APEX require heterologous expression and provide limited quantitative information. Cross-linking mass spectrometry (XL-MS) can identify interaction sites but typically requires specialized analysis pipelines.

TIE-UP-SIN (targeted interactome experiment for unknown proteins by stable isotope normalization) addresses these limitations by combining reversible formaldehyde crosslinking to stabilize PPIs in vivo, uniform ¹⁵N metabolic labeling for accurate quantification, and a stringent triple-control design to distinguish specific from nonspecific interactors. The method builds upon the SPINE approach [1], extending it with isotopic labeling for robust, ratio-based quantification. Unlike classical SILAC, which is challenging in prototrophic bacteria, TIE-UP-SIN uses global ¹⁵N labeling that is compatible with minimal media and does not require auxotrophic strains.

The protocol is particularly suited for bacterial systems where genetic manipulation is feasible and can be applied to any protein that can be tagged with an affinity purification tag. While demonstrated here with the Twin-Strep-tag system in Bacillus subtilis, the approach can be adapted to other tags and organisms.

Materials and reagents

Biological materials

1. Wildtype strain of the organism of choice, e.g., Bacillus subtilis BSB1 wildtype strain [2] (BaSysBio-Consortium)

2. Bait strain (wildtype strain with C-terminal Twin-Strep-tag fusion to the bait gene), e.g., Bacillus subtilis bait strain with chromosomally integrated C-terminal Twin-Strep-tag fusion [3]

Reagents

1. BioExpress^® bacterial cell media (unlabeled) (10× concentrate) (Cambridge Isotope Laboratories, catalog number: CGM-1000-U)

2. BioExpress^® bacterial cell media (U-15N, 98%) (10× concentrate) (Cambridge Isotope Laboratories, catalog number: CGM-1000-N)

3. Paraformaldehyde extra pure (Carl Roth, catalog number: 0335.1)

4. N-2-Hydroxyethylpiperazine-N′-2-ethane sulphonic acid (HEPES) (Carl Roth, catalog number: 9105.3)

5. Water, Baker HPLC analyzed, J.T. Baker^TM (Fisher Scientific, catalog number: 10546602)

6. Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771.1)

7. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4509)

8. Magnesium chloride hexahydrate (MgCl₂) (Supelco, catalog number: 105833025)

9. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.2)

10. Pierce^TM universal nuclease for cell lysis (Thermo Fisher Scientific, catalog number: 88700)

11. Micro BCA^TM Protein Assay kit (Thermo Fisher Scientific, catalog number: 23235)

12. Tween^® 20 (Sigma-Aldrich, catalog number: P7949)

13. MagStrep^® Strep-Tactin^® XT beads (IBA Lifesciences, catalog number: 2-5090-010)

14. Biotin (IBA Lifesciences, catalog number: 2-1016-002)

15. Calcium chloride hexahydrate (CaCl₂) (Sigma-Aldrich, catalog number: 12074)

16. Sera-Mag^TM SpeedBead carboxylate-modified magnetic particles (Cytiva, catalog number: 65152105050250)

17. Acetonitrile hypergrade for LC-MS (Merck, catalog number: 1000291000)

18. Lys-C, mass spec grade (Promega, catalog number: VA1170)

19. Sequencing-grade modified trypsin (Promega, catalog number: V5111)

20. 2xiRT kit (Biognosys, catalog number: 1900615)

21. Trifluoroacetic acid, LC-MS grade (TFA) (Thermo Fisher Scientific, catalog number: 85183)

22. Glycerol (Sigma-Aldrich, catalog number: 49767)

Solutions

1. BioExpress bacterial cell media unlabeled (see Recipes)

2. BioExpress bacterial cell media ¹⁵N-labeled (see Recipes)

3. Formaldehyde crosslinking buffer (see Recipes)

4. Disruption buffer (see Recipes)

5. Benzonase dilution buffer (see Recipes)

6. Buffer W (see Recipes)

7. 20 mM HEPES pH 8.0 + 2% Tween-20 (see Recipes)

8. Elution buffer XT (see Recipes)

9. 200 mM HEPES pH 8.0 + 4 mM CaCl₂ (see Recipes)

10. 100 mM HEPES pH 8.0 + 2 mM CaCl₂ (see Recipes)

11. Digestion buffer (see Recipes)

Recipes

1. BioExpress bacterial cell media unlabeled

Reagent	Final concentration	Volume
BioExpress^® bacterial cell media (unlabeled) (10× concentrate)	1×	60 mL
Water, Baker HPLC analyzed	n/a	540 mL
Total	n/a	600 mL

Prepare fresh BioExpress^® bacterial cell media (unlabeled) for each experiment by diluting it 1:10 with Baker HPLC water to the required cultivation volume. Filter-sterilize (0.2 μM pore size) the diluted media and add antibiotics as needed for plasmid-containing strains. Maintain sterile conditions throughout all preparation steps.

2. BioExpress bacterial cell media ¹⁵N-labeled

Reagent	Final concentration	Volume
BioExpress^® bacterial cell media (U-15N, 98%) (10× concentrate)	1×	60 mL
Water, Baker HPLC analyzed	n/a	540 mL
Total	n/a	600 mL

Prepare fresh BioExpress^® bacterial cell media (U-15N, 98%) for each experiment by diluting it 1:10 with Baker HPLC water to the required cultivation volume. Filter-sterilize (0.2 μM pore size) the diluted media and add antibiotics as needed for plasmid-containing strains. Maintain sterile conditions throughout all preparation steps.

3. Formaldehyde crosslinking buffer

Reagent	Final concentration	Quantity or volume
Paraformaldehyde extra pure	4%	2.4 g
50 mM HEPES pH 8.0	n/a	60 mL
Total	n/a	60 mL

Dissolve 2.4 g of paraformaldehyde in 50 mL of 50 mM HEPES, pH 8.0. Gently warm the buffer on a heated magnetic stir plate and stir using a magnetic stir bar. For safety, work under a fume hood and ensure the buffer does not boil during heating. Dissolution of paraformaldehyde takes some time. Adjust the pH to 8.0 with 1 M NaOH. While adding NaOH, the paraformaldehyde will fully dissolve. Afterward, bring the volume up to 60 mL with 50 mM HEPES pH 8.0 for a 4% (w/v) formaldehyde solution. Optionally, filter sterilize. Prepare the amount of formaldehyde crosslinking buffer required for the experiment. This buffer should always be prepared fresh before use.

4. Disruption buffer

Reagent	Final concentration	Volume
100 mM HEPES pH 8.0	20 mM	10 mL
Baker HPLC water	n/a	40 mL
Total	n/a	50 mL

The volume of the disruption buffer to prepare depends on the number of samples. This buffer can be stored protected from light for up to one year.

5. Benzonase dilution buffer

Reagent	Final concentration	Volume
1 M HEPES pH 8.0	20 mM	5 mL
1 M NaCl	20 mM	5 mL
1 M MgCl₂	2 mM	0.5 mL
Water, Baker HPLC analyzed	n/a	239.5 mL
Total	n/a	250 mL

6. Buffer W

Reagent	Final concentration	Volume
100 mM HEPES pH 8.0	20 mM	30 mL
100% Tween-20	1%	1.5 mL
Water, Baker HPLC analyzed	n/a	117 mL
Total	n/a	150 mL

This buffer can be stored protected from light for up to one year.

7. 20 mM HEPES pH 8.0 + 2% Tween-20

Reagent	Final concentration	Volume
100 mM HEPES pH 8.0	20 mM	2 mL
100% Tween-20	2%	0.2 mL
Water, Baker HPLC analyzed	n/a	7.6 mL
Total	n/a	10 mL

This buffer can be stored protected from light for up to one year.

8. Elution buffer XT

Reagent	Final concentration	Quantity or volume
Biotin	100 mM	1.22 g
20 mM HEPES pH 8.0	n/a	50 mL
Total	n/a	50 mL

Adjust the pH to 8.0. This buffer can be stored protected from light at 4 °C.

9. 200 mM HEPES pH 8.0 + 4 mM CaCl₂

Reagent	Final concentration	Volume
1 M HEPES pH 8.0	200 mM	1 mL
1 M CaCl₂	4 mM	0.02 mL
Water, Baker HPLC analyzed	n/a	3.98 mL
Total	n/a	5 mL

10. 100 mM HEPES pH 8.0 + 2 mM CaCl₂

Reagent	Final concentration	Volume
200 mM HEPES pH 8.0 + 4 mM CaCl₂	100 mM + 2 mM CaCl₂	2 mL
Water, Baker HPLC analyzed	n/a	2 mL
Total	n/a	4 mL

11. Digestion buffer

Reagent	Final concentration	Volume
100 mM HEPES pH 8.0 + 2 mM CaCl₂	50 mM HEPES + 1 mM CaCl₂	2 mL
Water, Baker HPLC analyzed	n/a	2 mL
Total	n/a	4 mL

Laboratory supplies

1. Cell culture flask, 100 mL (DWK Life Sciences, catalog number: 217712401)

2. Cell culture flask, 300 mL (DWK Life Sciences, catalog number: 217713903)

3. Filter tip, 20 μL (Sarstedt, catalog number: 70.3030.265)

4. Laboratory bottles, Duran^®, 100 mL (DWK Life Sciences, catalog number: 215-1514)

5. Laboratory bottles, Duran^®, 500 mL (DWK Life Sciences, catalog number: 215-1516)

6. Laboratory bottles, Duran^®, 1,000 mL (DWK Life Sciences, catalog number: 215-1517)

7. SafeSeal microcentrifuge tubes, 1.7 mL (Sorenson BioScience, catalog number: 11720)

8. SafeSeal SurPhob tips, 10 μL, sterile (Biozym, catalog number: VT0200)

9. SafeSeal SurPhob tips, 100 μL, sterile (Biozym, catalog number: VT0230)

10. SafeSeal SurPhob tips, 200 μL, sterile (Biozym, catalog number: VT0240)

11. SafeSeal SurPhob tips, 1,250 μL, sterile (Biozym, catalog number: VT0270)

12. Screw cap tube, 15 mL (Sarstedt, catalog number: 62.554.502)

13. Screw cap tube, 50 mL (Sarstedt, catalog number: 62.547.254)

14. Serological pipette, 5 mL (Sarstedt, catalog number: 86.1253.001)

15. Serological pipette, 10 mL (Sarstedt, catalog number: 86.1254.001)

16. Serological pipette, 25 mL (Sarstedt, catalog number: 86.1685.001)

17. Serological pipette, 50 mL (Sarstedt, catalog number: 86.1256.001)

18. Steritop^® 45 mm neck size, 0.22 μm pore size, PES (Millipore, catalog number: S2GPT05RE)

19. Tube, 13 mL (Sarstedt, catalog number: 62.515.006)

20. MS-vials (VWR, catalog number: 548-3018)

21. 0.1 mL micro-insert for vials with small opening, 31 × 5 mm, clear glass, first hydrolytic class, 15 mm top (VWR, catalog number: 548-00-20A)

22. Acclaim^TM PepMap^TM 100 C18 HPLC columns (Thermo Fisher Scientific, catalog number: 164946)

Equipment

1. Adventurer^TM Precision (Ohaus, catalog number: 30805891)

2. Centrifuge 5804 R (Eppendorf, catalog number: 5805000010)

3. Eppendorf Thermo Mixer^® C (Eppendorf, catalog number: 5382000015)

4. Fisherbrand^TM Mini-Centrifuge (Fisher Scientific, catalog number: 16617645)

5. Galaxi Mini Centrifuge (VWR, catalog number: 019511)

6. GFL-3005 Shaker (GFL Gesellschaft Fuer LabortecTM 3005, catalog number: 10186650)

7. Heraeus B12 Function Line (Thermo Scientific, catalog number: 50042307)

8. Loopster digital (IKA, catalog number: 0004016000)

9. Micro Star 17 (VWR, catalog number: 521-1646)

10. Mini Laboratory Pump, VP86 (VWR, catalog number: 181-0067)

11. Mixer Mill MM400 (Retsch, catalog number: 20.715.0001)

12. New Brunswick Innova^® 44 Incubator Shaker (Eppendorf, catalog number: M1282-0002)

13. Orbitrap Exploris^TM 480 (Thermo Fisher Scientific, catalog number: BRE725539)

14. Pipet Controller accu-jet^® pro (Brand, catalog number: 26300)

15. Rainin Pipet-Lite^TM LTS (Mettler Toledo, catalog numbers: 17014393, 17014392, 17014391)

16. Rocking platform shaker (VWR, catalog number: 444-0145)

17. Safety Cabinets Safe 2020 (Thermo Fisher Scientific, catalog number: 51026637)

18. Sonorex Super RK 31 ultrasonic bath (Bandelin, catalog number: 329)

19. Thermo Top^® (Eppendorf, catalog number: 5308000003)

20. Transferpette^® S, Typ variable (Brand, catalog numbers: 705869, 705870, 705872, 705874, 705878, 705880)

21. UltiMate^TM 3000 RSLC (Thermo Fisher Scientific, catalog number: 6041.0001)

22. V-1200, Vis-Spectrophotometer (VWR, catalog number: 634-6000)

23. VMS-A, magnetic hotplate stirrer (VWR, catalog number: 442-0185)

24. Retsch grinding jar (Retsch, catalog number: 01.462.0231)

Software and datasets

1. Spectronaut^® version 17 or higher (Biognosys AG) (requires license)

2. DIA-NN version 2.2.0 or higher (requires no license) [4]

3. Chosen organisms protein database (e.g., from UniProt; https://www.uniprot.org)

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: Jan 21, 2026

接收日期: Mar 13, 2026

在线发布日期: Mar 30, 2026

出版日期: Apr 20, 2026

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如何引用

Schedlowski, M., Michalik, S., Hoffmüller, T., Harms, M., Steil, L., Surmann, K., Hentschker, C., Salazar, M. G., Völker, U. and Reder, A. (2026). TIE-UP-SIN: A Method for Enhanced Identification of Protein–Protein Interactions. Bio-protocol 16(8): e5663. DOI: 10.21769/BioProtoc.5663.