Exploris 480 mass spectrometer

The proteome and phosphoproteome fractions were analysed by LC/MS/MS using a Thermo Ultimate 3000 nLC coupled to an Exploris480 mass spectrometer (Thermo Scientific). Samples were injected onto an Ion Opticks Aurora C18 column (75 μm internal diameter × 15 cm, 1.6 μm particle size) and separated over a 70- or 100-min method. The gradient for separation consisted of 5–42% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% acetonitrile. The Exploris480 was operated in turboTMTpro mode with a cycle time of 3 s. Resolution for the precursor scan (m/z 375–1,400) was set to 60,000 with a automatic gain control (AGC) target set to standard and a maximum injection time set to auto. MS2 scans (30,000 resolution) consisted of higher collision dissociate set to 38; AGC target set to 300%; maximum injection time set to auto; isolation window of 0.7 Da; fixed first mass of 110 m/z.

Twenty proteins were screened through bioinformatics analyses, and further verified with PRM. The digested peptides were dissolved in 0.1% formic acid (mobile phase A) and analyzed using PRM method on Exploris 480™ mass spectrometer (Thermo) coupled to an Easy-NLC 1200 UPLC-system (ThermoFischer). The detection and analysis of peptide parent ions and their secondary fragments were performed in the Orbitrap analyzers. Data was acquired with Targeted Mass modes. Higher-energy collision dissociation (HCD) was set at normalized collision energy of 27%. Mass spectrometer settings were, for full MS acquisitions, mass-to-charge ratio (m/z) range set at 395-815 m/z, resolution set at 60,000, automatic gain control (AGC) set at 300%, maximum ion injection time of 100 ms; for MS/MS acquisitions, resolution set at 15,000, automatic gain control (AGC) set at 75%, maximum ion injection time set to 100 ms, isolation window set to 1.6 m/z.

R. capsulatus strains were cultured anaerobically until a total OD₆₆₀ of 3 was achieved. Cell samples were prepared by three centrifugation steps and two washing steps with phosphate buffer (3.6 g Na₂HPO₄ × 2 H₂O and 2.6 g KH₂PO₄ per liter distilled H₂O). For protein extraction frozen cell pellets were re-suspended in 2% sodium lauroyl sarcosinate (SLS) and heated for 15 min at 90°C. Proteins were reduced with 5 mM Tris(2-carboxyethyl) phosphine (Thermo Fischer Scientific) at 90°C for 15 min and alkylated using 10 mM iodoacetamide (Sigma Aldrich) at 20°C for 30 min in the dark. Proteins were precipitated with a sixfold excess of ice-cold acetone, followed by two methanol washing steps. Dried proteins were reconstituted in 0.2% SLS and the amount of proteins was determined by bicinchoninic acid protein assay (Thermo Scientific). For tryptic digestion, 50 µg protein was incubated in 0.5% SLS and 1 µg of trypsin (Serva) at 30°C overnight. Desalted peptides were then analyzed by liquid chromatography-mass spectrometry using an Ultimate 3000 RSLC nano connected to an Exploris 480 mass spectrometer (both Thermo Scientific). Label-free quantification of data -independent acquisition raw data was done using DIA-NN. Further details on sample processing, analytical set up, and bioinformatics analysis are described in the Supplement.

To identify differentially enriched proteins in the supernatants of Btt and Bt407- cultures, we performed high resolution LC-MS/MS analysis by using an EASY-nLC 1200 (Thermo Fisher) coupled to an Exploris 480 mass spectrometer (Thermo Fisher). We separated peptides on 20 cm frit-less silica emitters (CoAnn Technologies, 0.75 µm inner diameter), packed in-house with reversed-phase ReproSil-Pur C₁₈ AQ 1.9 µm resin (Dr Maisch) and kept the column constantly at 50°C. We acquired the mass spectra in data-dependent acquisition mode as outlined in Sindlinger et al. [19 (link)]. We processed the raw data, by using the MaxQuant software version 2.0.3.0 [20 (link)]. MS/MS spectra were assigned to a custom Btt proteome assembly (Dr Heiko Liesegang, Institute of Microbiology and Genetics, University of Göttingen, unpublished) with default settings and match between runs; LFQ and iBAQ options were enabled. For further downstream analysis we used R [21 ]. After log₂ transformation, we imputed missing LFQ intensities based on quantile regression using imputeLCMD. To test for differential expression, we used LIMMA [22 (link),23 (link)]. Raw data were uploaded to the JPOST repository [24 (link)].

Lyophilized peptides were dissolved
in 0.1% formic acid (FA) and analyzed by online C18 nano-HPLC MS/MS
with a system consisting of an Ultimate3000nano gradient HPLC system
(Thermo, Bremen, Germany), and an Exploris480 mass spectrometer (Thermo).
Fractions were injected onto a cartridge precolumn (300 μm ×
5 mm, C18 PepMap, 5 μm, 100 A) and eluted via a homemade analytical
nano-HPLC column (50 cm × 75 μm; Reprosil-Pur C18-AQ 1.9
um, 120 A (Dr. Maisch, Ammerbuch, Germany)). The gradient was run
from 2 to 36% solvent B (20/80/0.1 water/acetonitrile/FA v/v) in 120
min. The nano-HPLC column was drawn to a tip of ∼10 μm
and acted as the electrospray needle of the MS source. The mass spectrometer
was operated in data-dependent MS/MS mode for a cycle time of 3 s,
with a HCD collision energy at 36% and recording of the MS2 spectrum
in the Orbitrap, with a quadrupole isolation width of 1.2 Da. In the
master scan (MS1), the resolution was 120,000, the scan range 350–1600,
at standard AGC target @maximum fill time of 50 ms. A lock mass correction
on the background ion m/z = 445.12
was used. Precursors were dynamically excluded after n = 1 with an exclusion duration of 45 s, and with a precursor range
of 20 ppm. Charge states 2–5 were included. For MS2, the first
mass was set to 110 Da, and the MS2 scan resolution was 45,000 at
an AGC target of 200% with a maximum fill time set to auto.

To achieve high sensitivity, a nanoLC system (Vanquish Neo UHPLC—part of Thermo Scientific) using an Acclaim PEPMap C18 column (25cm × 75 µm ID, Thermo Scientific, Waltham, MA, USA) was coupled online to an Exploris 480 mass spectrometer (Thermo Fisher Scientific). Peptides were separated using a 130 min binary gradient of water and acetonitrile containing 0.1% formic acid. Data-independent acquisition (DIA) was performed using a MS1 full scan (400 m/z to 1200 m/z) followed by 60 sequential DIA windows with an overlap of 1 m/z and the window placement optimization option enabled. Full scans were acquired with a 120,000 resolution, an automatic gain control (AGC) of 3 × 10⁶, and a maximum injection time of 50 ms. Afterwards, 60 isolation windows were scanned with a resolution of 30,000 and an AGC of 8 × 10⁵; the maximum injection time was set to auto to achieve the optimal cycle time. Collision-induced dissociation fragmentation was induced with 30% of the normalized HCD collision energy.
The data were analyzed with the software DIA-NN (version 1.8.1) by using a predicted library generated from an in silico digested Citrus sinensis (UP000027120) Uniprot reference database involving cuts at K* and R* with two missed cleavages allowed and a minimal peptide length set at 6. The false discovery rate for peptide and protein identification was set at 0.01%.