Protein calibration standard 2

S100A8, S100A9 and TGFβ1 and calcium ions (CaCl₂) were mixed at an equimolar concentration (11.3 μM) in an aqueous solution. Each mixture was incubated overnight at 37°C prior to MALDI-TOF/MS analysis. S100A8, S100A9 and TGFβ1 mixtures were diluted 1/10 in 50 μL of 0.1% TFA aqueous solution, after which 5 microliters of each dilution were added to 5 μl of Sinapinic acid (SA); 1 μl of the resulting mixture was deposited in duplicate on the steel sample holder and allowed to dry. External mass calibration (Protein Calibration Standard II, BrukerDaltonics) was based on monoisotopic values of [M + H] + of Trypsinogen, Protein A, Albumin-Bovine (BSA), at m/z 23982, 44613, 66500, respectively. MALDI-TOF/MS measurements were performed using an Ultraflex II MALDI-TOF instrument (BrukerDaltonics), operating in linear positive ion mode. Ions were formed using a pulsed UV laser (λ = 337 nm) beam. The instrumental conditions were: IS1 = 25 kV; IS2 = 23.35 kV; delay time = 70 nsec. All chemicals and solvents were purchased from Sigma-Aldrich (Sigma Aldrich). One spectrum was collected for each sample, an average of 1,000 laser shots being obtained from the respective two replicate spots.

Matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) analysis were performed on a Microflex II mass spectrometer (Bruker Daltonics, Germany). Depending on the concentration, samples were used directly or concentrated following the ZipTip C4 Millipore protocol. A saturated solution of sinapinic acid made in acetonitrile-water-trifluoroacetic acid (50:50:0.1) was used as the matrix. Samples were treated according to the dry droplet method; mixtures were allowed to dry at room temperature. Deposits were re-crystallized by the addition of matrix. Data were acquired in a positive linear mode; depending on the mass analyzed, the range was set from 20 to 50 kDa or from 50 to 85 kDa, and pulsed ion extraction was fixed respectively to 350 ns or 500 ns. External mass calibration was done just before the acquisition of the sample using protein calibration standard II (Bruker Daltonics, Germany). Mass spectra were examined in Flex Analysis software, no smoothing or baseline subtraction was applied. For each version of SipB protein (wild-type or variant) and for each condition of production (with or without IacP) independent preparations of purified protein and mass spectrometry analysis were made at least twice.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed using an ultra-flex treme mass spectrometer (Bruker, Leiderdorp, The Netherlands). Proteins were desalted using Micro Bio-Spin P-6 columns (Bio-Rad, Veenendaal, The Netherlands), and samples were prepared by the dried droplet method on a 600 µm Anchor Chip target (Bruker, Leiderdorp, The Netherlands), using 8 mg mL⁻¹ 2,5-dihydroxyacetophenone, 1.5 mg mL⁻¹ diammonium hydrogen citrate, 25% (v/v) ethanol and 3% (v/v) trifluoroacetic acid as matrix. Spectra was derived from ten 500-shot (1000 Hz) acquisitions taken at non-overlapping locations across the sample. Wide mass-range measurements were made in the positive linear mode, with ion source 1, 25.0 kV; ion source 2, 23.3 kV; lens, 6.5 kV; pulsed ion extraction, 680 ns. Detailed analyses of glycoproteins in the ~ 31–51 kDa range were done with ion source 1, 20.0 kV; ion source 2, 18.4 kV; lens, 6.2 kV; pulsed ion extraction, 450 ns, and spectra were derived from ten 1000-shot (1000 Hz) acquisitions. Protein Calibration Standard II (Bruker, Leiderdorp, The Netherlands) was used for external calibration.

Protein masses were determined on purified solution samples. To this end, the proteins in 8 M urea containing buffer were loaded onto a G-25 column (Cytiva, Marlborough, MA, USA) and eluted with 10 mM ammonium acetate. After a concentration step using Zip Tip C4 (Merck Millipore Darmstadt, Germany), 1 μL of protein at ~1.5 mg/mL was mixed with 1 μL of sinapinic acid matrix solution in 0.3% TFA/CH₃CN (50:50 v/v). One μL of the mix was spotted on the target and analyzed by MALDI-TOF on a Ultraflex III spectrometer (Bruker Daltonics, Wissembourg, France) controlled by the Flexcontrol 3.0 package (Build 51) and operated in the linear mode, using a maximum accelerating potential of 25 kV and a 20,000–100,000 m/z range (LP_66kDa method). The laser frequency was fixed to 100 Hz and ~1000 shots per sample were cumulated. Four external standards (Protein Calibration Standard II, Bruker Daltonics) were used to calibrate each spectrum to a mass accuracy within 100 ppm. Peak picking was performed using the FlexAnalysis 3.0 software with an adapted analysis method. Parameters used were the centroid peak detection algorithm, S/N threshold fixed to 5 and a quality factor threshold of 30.