A Membrane‐Permeable and Immobilized Metal Affinity Chromatography (IMAC) Enrichable Cross‐Linking Reagent to Advance In Vivo Cross‐Linking Mass Spectrometry

Abstract Cross‐linking mass spectrometry (XL‐MS) is an attractive method for the proteome‐wide characterization of protein structures and interactions. Currently, the depth of in vivo XL‐MS studies is lagging behind the established applications to cell lysates, because cross‐linking reagents that can penetrate intact cells and strategies to enrich cross‐linked peptides lack efficiency. To tackle these limitations, we have developed a phosphonate‐containing cross‐linker, tBu‐PhoX, that efficiently permeates various biological membranes and can be robustly enriched using routine immobilized metal ion affinity chromatography. We have established a tBu‐PhoX‐based in vivo XL‐MS approach that enables cross‐links in intact human cells to be identified in high numbers with substantially reduced analysis time. Collectively, the developed cross‐linker and XL‐MS approach pave the way for the comprehensive XL‐MS characterization of living systems.

The supernatant was evaporated in vacuo and the crude product was purified by column chromatography (Heptane/EtOAc 0 to 3:1) to obtain compound 2 in a solid state (

Synthesis of 5-(di-tert-butoxyphosphoryl) isophthalic acid
Dimethyl 5-(di-tert-butoxyphosphoryl) isophthalate (4.0 g, 10.13 mmol, 1 equiv.) was dissolved in tetrahydrofuran in a round bottom flask. Next LiOH was added (0.74 g, 41.6 mmol, 3 equiv.) to a round bottom flask in a single portion. Then water was added to dissolve lithium hydroxide in the reaction mixture and stirred for 4-5 h. The reaction mixture pH was adjusted to 2-3 with acetic acid and the aqueous phase was extracted with EtOAc (2x), the combined organic phases were dried over Na2SO4, filtered and concentrated in vacuo to give the pure product dimethyl 5-(di-tert-butoxyphosphoryl) isophthalic acid (2.5 g, 6.98 mmol) which equals a yield of 67.3 %.

Synthesis of tBu-PhoX (tert-Butyl Disuccinimidyl Phenyl Phosphonate)
5-(di-tert-butoxyphosphoryl) isophthalic acid (2.30 g, 6.41 mmol, 1 equiv.) was dissolved in methylene chloride, 1.81 g (17.97 mmol, 2.8 equiv.) of trimethylamine was added into the solution and then followed by addition of 3.52 g TFA. Afterwards, NHS-TFA (16.6 mmol, 2.6 equiv.) was added into the solution. The reaction mixture was stirred for 14 h at room temperature. The crude reaction was purified by column chromatography using a mixture of ethyl acetate and methanol to give pure product of tBu-Phox (1.8g, 2.3 mmol).

Materials for biochemical experiments
Chemicals for the following method sections were acquired from Sigma-Aldrich, Carl Roth or Merck unless otherwise stated.

Cell culture
HEK293T cells were cultured in Eagle's minimal essential medium (DMEM) from Gibco (DMEM, high glucose, GlutaMAX™) supplemented with 10 % (v/v) fetal bovine serum (Gibco) at 37 ºC with 5% CO2. E. coli were cultured in modified M9 minimal medium [1] and B. subtilis were cultured in C minimal medium [2] to the middle of the log growth phase at approximately OD 600 nm 0.5. Cells were harvested, washed with PBS and concentrated to OD 600 nm 100.

Isolation of mouse heart mitochondria
Hearts of adult mice were isolated and washed twice with PBS, pH 7.4 (Gibco). TH buffer (300 mM Trehalose, 10 mM KCl, 10 mM HEPES, pH 7.4) supplemented with 0.1 mg/ml BSA was used for homogenization. Homogenization was performed 25 times at 1,000 rpm on ice. Cell debris was removed by centrifugation at 400 g for 10 min. The supernatant was collected and subjected to a second centrifugation step at 800 g for 10 min to further remove cell debris. The supernatant was collected and mitochondria in the supernatant were pelleted at 11,000 g for 10 min. Mitochondria were washed three times with TH buffer without BSA. All centrifugation steps were performed at 4°C.

Cross-link enrichment
Digested peptide mixture was treated with 0.5% TFA for 2 h at room temperature to remove the t-butyl groups. Sample was then diluted to 80% ACN/0.1% TFA by adding 100% ACN to prepare for enrichment. Agarose Ni-NTA beads were first de-chelated by incubation in 100 mM EDTA for 30 min, and then chelated by incubation in 10 mM FeCl3 for 30 min. Next, peptides were added to conditioned agarose Fe-NTA beads using a sample-to-bead ratio of 10:1 (v/v), and incubated on a shaker for 30 min. Beads were collected, loaded onto C8 StageTip (packed with YMC*GEL C8-HG Packing Material (YMC)), and washed with 80% ACN/0.1% TFA 3 times.
Crosslinker-modified peptides were eluded from Fe-NTA beads to C8 StageTip with 500 mM potassium phosphate buffer (192 mM monobasic potassium phosphate and 308 mM dibasic potassium phosphate). Peptides bound to the C8 material were then washed with 0.1% FA, followed by elution with 50% ACN/0.1% FA. Eluted peptides were dried in SpeedVac for subsequent analysis.

Size Exclusion Chromatography (SEC) fractionation
Enriched cross-linker-modified peptides were fractionated by SEC using a SuperdexTM 30 Increase 3.2/300 column (GE Healthcare) on an Agilent 1260 Infinity II system. SEC was performed with a 90 min run using an isocratic flow of 0.05 ml/min. Mobile phase was 30% ACN/0.1% TFA. Five early SEC fractions were collected, dried under SpeedVac and subjected to LC-MS analysis.

LC-MS analysis
LC-MS analysis was performed using an UltiMate 3000 RSLC nano LC system coupled on-line to an Orbitrap Fusion mass spectrometer or an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). High field asymmetric waveform ion mobility spectrometry (FAIMS) (Thermo Fisher Scientific) was enabled. Reversed-phase separation was performed using a 50 cm analytical column (in-house packed with Poroshell 120 EC-C18, 2.7 µm, Agilent Technologies) with 120 min or 180 min gradients for single-shot experiments or 180 min gradients for SEC fractions. Cross-link acquisition was performed using a LC-MS2 method. The following parameters were applied: MS resolution 120,000; MS2 resolution 30,000; charge state 3-8 enable for MS2 (unless otherwise stated); HCD energy 30, FAIMS internal stepping -50/-60/-70 V (unless otherwise stated).

Data analysis
Data analysis was performed using pLink 2.3.9 [3] with the following parameters: minimum

Figure S4
Cell viability assay of HEK293T cells using trypan blue staining. The viability of the cells were measured before cross-linking (0 min) and after 5, 15, 30 and 60 min cross-linking using 2 mM tBu-PhoX. The experiment was performed in triplicates.

Figure S5
Number of spectra and unique peptides for each peptide type in tBu-PhoX cross-linked cells.

Figure S6
Distribution of cross-links versus mono-links in different SEC fractions. A) Percentage and number of cross-links and mono-links in different SEC fractions. B) Cosine similarity of crosslinks identified in different SEC fractions.

Figure S7
In vivo cross-linking of intact HEK293T cells using tBu-PhoX. A) Euler diagram shows the overlap of cross-link identifications between SEC fractions and the single-shot LC-MS measurement. B) GO terms (BP, CC, MF) from identified inter-protein cross-links using spectrum count as a quantitative measurement.

Figure S8
Visualization of cross-links on high-resolution structures of selected protein complexes.