Dionex ultimate 3000 rslc

Separation was achieved on an UHPLC system Dionex Ultimate 3000RSLC (ThermoFisher Scientific, Inc., Waltham, MA, USA) with reversed phase column Kromasil Eternity XT C18 (1.8 µm, 2.1 × 100 mm) column maintained at 40 °C. The binary mobile phase consisted of A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile. The run time was 33 min. The following gradient was utilized: the mobile phase was held at 5% B for 1 min, gradually turned to 30% B over 19 min, increased gradually to 50% B over 5 min, increased gradually to 70% B over 5 min, and finally increased gradually to 95% over 3 min. The system was then turned to the initial condition of 5% B and equilibrated over 4 min. The flow rate and the injection volume were set to 300 µL/min and 1 µL, respectively. The effluents were connected on-line with a Q Exactive Plus Orbitrap mass spectrometer where the compounds were detected [22 ].

The shell samples collected from sucrose fractions were washed with PBS buffer and were treated for mass spectrometry analysis^{24 (link)}. Data-dependent LC−MS/MS analysis was performed on a QExactive quadrupole-Orbitrap mass spectrometer coupled to a Dionex Ultimate 3000 RSLC nano-liquid chromatograph (Thermo Fisher, UK) installed with Xcalibur software version 4.1. A Mascot Generic File, created by Progenesis QI (Version 3.0, Nonlinear Dynamics, Newcastle upon Tyne, UK), was searched against the H. neapolitanus carboxysome protein database from UniProt.

Protein digests were reconstituted in 0.1% formic acid (ThermoFisher Sci.) and analyzed using LC–MS/MS by loading onto a Dionex UltiMate® 3000 RSLC liquid chromatography (LC) system (ThermoFisher Sci.) using a PepMap RSLC C18 2um, 75 um × 50 cm EASY-Spray™ column (ThermoFisher Sci.). Peptides were separated using a 0.3 uL/min LC gradient comprising 2–90% mobile phase B in 0–150 min. Mobile phase A was 0.1% formic acid in Milli Q water and mobile phase B was 0.1% FA in acetonitrile (MilliporeSigma). Eluting peptides were directly injected into an Orbitrap Elite Velos mass spectrometer (ThermoFisher Sci.) and ionized using collision-induced dissociation (CID) in positive ion mode. A “top 15” data-dependent MS/MS analysis was performed (acquisition of a full scan spectrum followed by CID mass spectra of the 15 most abundant ions in the survey scan). The data was submitted to the MassIVE repository: ftp://massive.ucsd.edu/MSV000087132/ for public access.

The Pierce Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific, Massachusetts, USA) was used to determine peptide concentrations post on-bead digestion. 20 µg of tryptic digest pooled from biological samples, were analysed using HpHRP. This was performed using a Dionex UltiMate 3000 RSLC (Thermo Fisher Scientific, Massachusetts, USA) coupled with an Acclaim PA II column (1.0 mm × 15 cm, C18, 3 µm, 120 Å) (Thermo Fisher Scientific, Massachusetts, USA). Mobile phase A contained 20 mM ammonium hydroxide in water (pH 9.6) and Mobile phase B contained 20 mM ammonium hydroxide (pH 9.6) supplemented with 80% acetonitrile in water. Peptide separation was done at 50 µl/min using a 35-min gradient (4–40% B). 30 fractions were collected every 30 secs from 12.5 to 27.5 min and then pooled to generate 10 concatenated fractions as per the following pooling scheme: (F1 = [1, 11, 21]; F2 = [2, 12, 22]; F3 = [3, 13, 23]; F4 = [4, 14, 24]; F5 = [5, 15, 25]; F6 = [6, 16, 26]; F7 = [7, 17, 27]; F8 = [8, 18, 28]; F9 = [9, 19, 29], F10 = [10, 20, 30]). Pooled fractions were vacuum dried using a CentriVap (Labconco, Missouri, USA) and stored at − 80 °C until further analysis.

For the LC-MS metabolomic analysis, the sample extraction was performed as described previously [81 (link),82 (link)]. Briefly, 5 × 10⁷ cells were used for each sample. Cells were rapidly cooled in a dry ice/ethanol bath to 4°C, centrifuged at 1,300g, 4°C for 10 minutes, washed with 1× PBS, and resuspended in extraction solvent (chloroform:methanol:water, 1:3:1 volume ratio). Following shaking for 1 hour at 4°C, samples were centrifuged at 16,000g at 4°C for 10 minutes, and the supernatant was collected and stored at −80°C. The analysis was performed using separation on 150 × 4.6 mm ZIC-pHILIC (Merck, Kenilworth, NJ) on Dionex UltiMate 3000 RSLC (Thermo Fisher Scientific Waltham, MA) followed by mass detection on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Waltham, MA) at Glasgow Polyomics.

Peptides were resuspended in resuspension buffer (2% acetonitrile, 0.1% triflouroacetic acid (Thermo Fischer Scientific, Waltham, MA, USA), 0.1% formic acid in MilliQ water) and a volume corresponding to ~ 1 μg peptide was analyzed by nanoLC-MS/MS (Thermo Scientific Dionex Ultimate 3000 RSLC) coupled in-line to a Thermo Scientific Q Exactive HF mass spectrometer. The peptide separation was accomplished using a precolumn setup (Acclaim PepMap 100 C18 2 cm 100 μm precolumn; 75 μm 75 cm main column) (Thermo Fischer Scientific, Waltham, MA, USA) and a 35-min gradient from 10% buffer B (99.9% acetonitrile) to 35% buffer B and the buffer A being 99.1% MilliQ with 0.1% formic acid. The mass spectrometer was set to acquire MS1 data from m/z 375–1500 at R = 60 k and MS2 at R = 30 k allowing up to 20 precursor ions per MS1 scan.