TITLE: A proximity proteomics pipeline for subcellular proteome and protein interaction mapping
AUTHORS: Xiaofang Zhong, qiongyu.li, Ruth Hüttenhain

[DESCRIPTION]
Proximity labeling (PL) coupled with mass spectrometry has emerged as a powerful technique to map proximal protein interactions in living cells. Large-scale sample processing for proximity proteomics necessitates a high-throughput workflow to reduce hands-on time and increase quantitative reproducibility. To address this issue, we developed a scalable and automated PL pipeline, including generation and characterization of monoclonal cell lines, automated enrichment of biotinylated proteins in a 96-well format, and optimization of the quantitative mass spectrometry (MS) acquisition method. Combined with data-independent acquisition (DIA) MS, our pipeline outperforms manual enrichment and data-dependent acquisition (DDA) MS regarding reproducibility of protein identification and quantification. We apply the pipeline to map subcellular proteomes for endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, using serotonin receptor (5HT2A) as a model, we investigated agonist-induced dynamics in protein-protein interactions. Importantly, the approach presented here is universally applicable for PL proteomics using all biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.

[STEPS]
SECTION: Cell culture
1. Seeding 3-4 million cells in a 10-cm dish or 500K cells in a 6-well plate on Day One. The cells will be ~70% confluent after 48 hours. 
SECTION: Cell lysis
10. Lyse cells in 1ml RIPA buffer supplement with protease inhibitors, antioxidants, and DTT
SECTION: Automated enrichment protocol for biotinylated proteins in Kingfisher
17. For the automated biotinylation enrichment protocol, the Kingfisher Flex system (Thermo Fisher) is programmed to simultaneously process a maximum of 96 samples. This protocol below includes two parts, where part 1 (Plate 1-3) is for washing magnetic streptavidin beads and binding of the biotinylated proteins to beads, and part 2 (Plate 4-12) is for washing and collecting beads prior to Lys-C/trypsin digestion. The enrichment protocol is conducted in the cold room using deep-well plates. 
SECTION: Protein digestion
20. Reduce proteins by adding 5mM TCEP (final concentration, add 2ul of a 500mM TCEP solution) and incubate by shaking at 1000rpm @37 degrees for 30min.
SECTION: C18 desalting
26. The peptide samples are desalted using C18 96-well plate (BioPureSPE, HNS S18V-20mg, the Nest group) 
SECTION: APEX proximity labeling
7. Invert the dish/plate and pour out the medium.
SECTION: APEX proximity labeling
8. Immediately wash the cells 3x with ice-cold quencher solution (PBS+ 5mM Trolox, 10 mM sodium azide, 10 mM sodium ascorbate) for 1min (8mL for 10cm dish or 1mL for 6-well plate). Aspirate or invert to remove.
SECTION: APEX proximity labeling
9. Collect the cells in fresh quencher solution (8ml for 10cm plate or 1mL for 6-well plate) and pellet the cells by centrifugation at 3000g for 10min at 4 oC, remove supernatant and continue to cell lysis or freeze cell pellets in dry-ice for storage
SECTION: Cell lysis
11. Perform a freeze-thaw cycle on dry ice and thaw in a 37 °C water bath

SECTION: Cell lysis
12. Sonicate samples (5 seconds, 15% Duty Cycle x 2)

SECTION: Cell lysis
13. Transfer samples to 1.5ml Eppendorf tubes
SECTION: Cell lysis
14. Centrifuge samples at 13,000xg for 10min at 4 °C and save the supernatant. 

SECTION: Cell lysis
15. Take a small amount (25 uL) of the lysate for WB analysis

SECTION: Cell lysis
16. Quantify protein concentration (supernatant) using 600nM Pierce Assay Kit with Detergent Compatibility Reagent (optional)
18. 1st day, protocol: Biotinylation APEX-MS part 1
19. 2nd day, protocol: Biotinylation APEX-MS part 2
18.1. Plate 1 (1 mL, stock)
a. RIPA buffer plate 1
b. Add 80 uL of streptavidin beads to each well of 1 mL of RIPA buffer
18.3. Plate 3 (1 mL, fresh lysis)
a. Sample binding plate
b. All samples from lysis are added to the plate
c. Beads are transferred to this plate after 2x RIPA wash and left in this plate for overnight binding
d. Tip comb is left in this plate at the end of the protocol
19.1. Plate 4-6 (1 mL, stock)
a. RIPA buffer plates 4-6
b. Continued from Plate 3
c. Beads with bond samples are collected from Plate 3 and transferred to these plates for 3x RIPA wash
19.2. Plate 7 (1 mL, stock)
a. KCl solution wash plate (1M KCl)
b. Stock solution prepared and can be stored for a long time
19.3. Plate 8 (1 mL, stock)
a. Na2CO3 solution wash plate (0.1M Na2CO3)
b. Stock solution prepared and can be stored for a long time
19.4. Plate 9 (1 mL, fresh)
a. 2M urea in 50mM Tris-HCl [pH=8.0] wash plate
b. 1 M Tris-HCl is ready
c. Urea prepared freshly each time
19.5. Plate 10-11 (1 mL, fresh)
a. 50mM Tris-HCl [pH=8.0] wash plates 10-11
b. Prepared from 1M Tris-HCl solution
19.6. Plate 12 (200 uL, fresh)
a. Digestion plate with 2M urea in 50mM Tris-HCl [pH=8.0] buffer
b. Beads and tip comb are left in this plate
18.2. Plate 2 (1 mL, stock)
a. RIPA buffer plate 2
b. 1 mL of RIPA buffer
SECTION: Protein digestion
21. Protein alkylation by adding iodoacetamide to 5mM (final concentration, add 2ul of a 500mM iodoacetamide solution) and incubate by shaking at 1000rpm @RT for 30min.
SECTION: Protein digestion
22. Quench IAA with 5mM DTT (final concentration, add 2ul of a 500mM iodoacetamide solution).
SECTION: Protein digestion
23. Add 2ul of trypsin (~1ug), and 1 ul of Lys-C (2ug/ul) and incubate O/N at 37 degrees under shaking at 1000 rpm. The on-bead digestion is set to 37 oC for 6h and RT for 12h. 
SECTION: Protein digestion
24. Following morning add additional 0.5 ug of trypsin and incubate an additional 2h at 37 °C .

SECTION: Protein digestion
25. Transfer supernatant to a new 96-well plate using magnetic rack, and acidify sample with 10% trifluoroacetic acid (TFA) to ~pH2 (~0.5% TFA final).
SECTION: C18 desalting
27. Wash the plate three times with 100 µL 80% acetonitrile (ACN)/0.1% TFA by centrifugation at 800 rpm for 1 min
SECTION: C18 desalting
28. Equilibrate the plate three times with 100 µL 2% ACN/0.1% TFA by centrifugation at 1200 rpm for 2 min
SECTION: C18 desalting
29. Load samples by centrifugation at 1600 rpm for 2 min
SECTION: C18 desalting
30. Re-load samples by centrifugation at 1600 rpm for 2 min
SECTION: C18 desalting
31. Wash the plate 3 times with 100 µL 2% ACN/0.1% TFA by centrifugation at 1600 rpm for 2 min
SECTION: C18 desalting
32. Elute twice with 55 µL 50% ACN/0.25% formic acid (FA) by centrifugation at 1600 rpm for 2 min
SECTION: C18 desalting
33. Dry the samples by vacuum centrifugation (~2h).
SECTION: APEX proximity labeling
5. Add 2mM H2O2-containing medium (DMEM+10% FBS) to the 10-cm dish or 6-well plate to have a final concentration of 1mM H2O2.
SECTION: APEX proximity labeling
3. Perform APEX proximity labeling on Day Three. 
2. Add doxycycline (final concentration of 1ug/mL) to induce cells for 24 hours on Day Two. 
SECTION: APEX proximity labeling
6. Allow the reaction to go for 45sec at room temperature.
SECTION: C18 desalting
34. Store the dried samples at -20 °C  or resuspend them in 20 uL 0.1% formic acid for mass spec analysis.

SECTION: APEX proximity labeling
4. Incubate cells with 500uM BP in complete medium pre-warmed to 37 oC for 30min.