Aminoguanidine hydrochloride

Microfluidic channels were prepared by mounting a square glass capillary
with inner diameter 0.8 × 0.8 mm² (VitroCom, USA)
on a microscopy slide using UV-curable adhesive (NOA 68, Norland Products
inc., USA). Pipette tips (Gel-loading Pipette Round Tips, VWR) were
inserted into and fixated with glue at both ends of the capillary.
The interior of the channels, as well as the coverslip glass surfaces
used for fluorescence microscopy and time-of-flight secondary ion
mass spectrometry (ToF-SIMS) analysis, was cleaned by immersion overnight
with Hellmanex III cleaning solution (2%, Hellma GmbH, Germany) and
rinsed with water (Milli-Q, Merck Life Science), followed by immersion
in sulfuric acid (2 M, Sigma-Aldrich 99.9%) for 1 h, followed by extensive
rinsing with water. The cleaned surfaces were rinsed with ethanol
(99.5%, Solveco, Sweden) and then silanized by immersion in 10% solution
of O-(propargyloxy)-N-(triethoxysilylpropyl)urethane
(90%, ABCR, Germany) in ethanol (99.5%, Solveco, Sweden) for 1 h.
The silanized capillaries and coverslips were rinsed with ethanol,
followed by water, and modified with AMP AMC-25-04 or α-mannose-PEG3-azide
(>95%, Sigma-Aldrich) or azide-fluor 488 (>90%, Sigma-Aldrich),
using
copper-catalyzed alkyne-azide cycloaddition (CuAAc, click-chemistry).^{49 (link)} This was followed by immersion of the surfaces
for 10 min in a click reaction solution containing 33 μM azidated
reactant, 17 mM aminoguanidine hydrochloride (Sigma-Aldrich), 75 μM
CuSO₄ (Sigma-Aldrich), 250 μM tris(3-hydroxypropyltriazolylmethyl)amine
(THPTA, Tokyo Chemical Industry Co., Ltd.), and 500 μM ascorbic
acid (Merk) diluted in PBS buffer (pH 7.4), whereupon the modified
surfaces were rinsed with water. After finishing the experiments,
the pristine glass surface of the channels was regenerated through
extensive cleaning with Hellmanex III overnight followed by immersion
in sulfuric acid (2 M, Sigma-Aldrich 99.9%).

Cu(I)-catalyzed click labeling of chemically-fixed microbial cells was performed on slides as described previously (Hatzenpichler et al., 2014 (link)). Briefly, fixed samples were immobilized on glass slides, dried in a 46°C hybridization oven, and dehydrated and permeabilized by placing slides for 3 min sequentially in 50, 80, and 96% ethanol. Then, 1.25 μL of 20 mM CuSO4, 2.50 μL of 50 mM tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine (THPTA) (baseclick GmbH, Germany), and 0.30 μL of alkyne dye (in DMSO) (Jena Bioscience, Germany) were mixed and allowed to react for 3 min at room temperature (RT) in the dark. In the meantime, 12.5 μL of freshly-prepared 100 mM sodium ascorbate (Sigma–Aldrich) and 12.5 μL of 100 mM aminoguanidine hydrochloride (Sigma–Aldrich) were added to 221 μL 1 × PBS (pH 7.4). Then, the dye premix was added to this solution, the tube inverted once and samples were covered by 30 μL of solution. Slides were transferred into a humid chamber and incubated in the dark at RT for 30 min. Afterward, slides were washed three times for 3 min each in 1 × PBS and then treated with an increasing ethanol series (3 min each in 50, 80, and 96% ethanol) and air-dried (Hatzenpichler et al., 2014 (link)). 1:1000 DNA stain, 4′, 6 diamidino-2-phenylindole (DAPI) solution (in PBS), was applied for 5 min and then slides were washed in cold MILLI-Q water (Millipore GmbH, Vienna, Austria). Samples were embedded with CitiFluor (Agar Scientific Ltd., Stansted, United Kingdom) if used immediately or stored at −20°C. Representative BONCAT pictures of a fecal sample incubated with rutin at 6 and 24 h and a negative control containing DMSO are shown in Supplementary Figure 1. Representative BONCAT picture of a fecal sample incubated with the glucose positive control is shown in Supplementary Figure 2. For FACS sorting, in-solution click labeling was performed immediately before FACS sorting. For click labeling, 300–500 μL fixed samples were centrifuged at 10,000 r/min for 10 min and re-suspended in 96% ethanol. The pellet was left for 3 min at RT and then centrifuged 10,000 r/min for 5 min. A master mix containing the dye solution was prepared as described above. Samples were suspended in 60–100 μL of solution and incubated in the dark at RT for 30 min. Afterward, samples were washed three times by centrifugation with 1x PBS (Hatzenpichler et al., 2014 (link)). Immediately before sorting, samples were filtered with a 35 μm nylon mesh using BD tubes 12 × 75 mm (BD, Germany).

Cell pellets were dissolved in a lysis buffer (0.3 M HEPES, 0.75 M NaCl, 0.1 M CHAPS) containing a 1× protease inhibitor cocktail (Roche, Basel, Switzerland). Cell pellets were sonicated using the following conditions: 10% Amplitude, 50% Duty cycle, 20 cycles, time: 80 s. Cell lysates were centrifuged at 15,000g at 4 °C for 30 min and the protein concentration of the supernatants was measured using a BCA protein assay kit (ThermoFisher Scientific). An equal amount of protein extract from the heavy and medium labeled extract was mixed. For the enrichment, the click-IT protein enrichment (ThermoFisher Scientific) was used using the vendor’s instructions with minor modifications. Initially, iodoacetamide was added to a final concentration of 55 mM and samples were incubated in the dark for 30 min. The samples were subsequently transferred to tubes containing the alkyne agarose resin. The click-IT reaction takes place in the presence of 200 mM Cu(II)SO₄ (Merck, Darmstadt, Germany), 160 mM Cu(I) ligand THPTA (supplied with the kit), 2 M aminoguanidine hydrochloride (Merck), and 2 M sodium ascorbate (supplied with the kit). The reaction mix was incubated for 2 h at 40 °C on a shaking platform while rotating. The beads were incubated with the one-step reduction/alkylation solution, which is the SDS wash buffer (contained in the kit) supplemented with 8 mM Tris(2‑carboxyethyl)phosphine (TCEP, Merck) and 33 mM 2-chloroacetamide (CAA, Merck), at 70 °C for 15 min. Samples were washed with SDS wash buffer, water, 5 times with guanidine wash buffer, and 5 times with acetonitrile wash buffer (20% acetonitrile). Resin was resuspended in the digestion buffer (0.1 M Tris pH = 8, 2 mM CaCl₂, 5% ACN). Trypsin/LysC was added to the samples in a 50:1 ratio and samples were digested for 18 h. Samples were acidified using formic acid and desalted using Sep-Pak cartridges. Cartridges were washed with neat acetonitrile and then with 60% ACN–0.1% trifluoroacetic acid (TFA, Biosolve, Dieuze, France). The cartridges were equilibrated in 0.1% TFA before sample loading. After sample loading, the cartridges were washed 3 times with 0.1% TFA. Finally, samples were eluted by using 60% ACN–0.1% TFA. Eluate was brought to dryness by Speedvac, and the peptides were resuspended in 0.1% TFA.