Autoflex 3 mass spectrometer

Two milligrams of AIR or hemicellulose fractions was digested with 4U of endo‐beta‐glucanase (Megazyme, Ireland), in sodium acetate buffer 0.01M pH 5 at 37 °C overnight. After addition of three volumes of cold 100% ethanol, the soluble fraction containing xyloglucan fragments was collected by centrifugation at 5.000 g. Supernatant was dried and then dissolved in 100 μL of 0.1% TFA. One microlitre was spotted on a MALDI‐TOF plate with DHB as matrix at 5 mg/mL in ACN ‐ TFA 0.1% (70:30, v:v). Spectra were recorded on a Voyager DE‐Pro from AB Sciex in positive reflector mode and accumulation of 3000 laser shots. MALDI‐TOF MS/MS experiments were performed using a Bruker Autoflex III mass spectrometer equipped with a frequency‐tripled Nd:YAG laser (355 nm), the Flex control 3.3 and Flex Analysis 3.3 software package (Bruker Daltonics, Bremen, Germany). For acquisitions in the tandem time‐of‐flight mode, the precursor ions were accelerated to a kinetic energy of 8 kV and selected in a timed ion gate. The fragment ions generated by laser‐induced decay (LID) of the precursor were accelerated to a kinetic energy of 19 kV in the LIFT cell was analysed after the ion reflector passage.

Once permethylated, purified glycans were solubilized in 20 µL of a 1:1 ratio methanol/DI water mix. A mix of 2 µL non-dilute and 2 µL N-glycans with 2  µL of 2,5-dihydroxybenzoic acid (LaserBio Labs, Sophia-Antipolis, France) matrix solution (10 mg/ml in 1:1 ratio methanol/DI water). Positive ion reflectron MALDI-TOF mass spectra were acquired using an Autoflex III mass spectrometer (Bruker Daltonics, Billerica, MA, USA). The acceleration and reflector voltage conditions were voltage 12 × 1977 V and 90% laser, and the spectra obtained by accumulation of 2,000 shots. The spectra were calibrated with an external standard (PepMix 4, LaserBioLabs, Sophia-Antipolis, France). To elucidate the glycan profiles of the sample, several spectra were obtained from different spots and the values averaged. To interpret the structures of the glycans corresponding to monisotopic masses after deisotoping of the spectra, the EXPAZY GlycoMod tool was used, along with GlycoWorkBench. Relative intensities of glycans were calculated to establish the glycan profile for each spectrum and mean values for the glycan intensities with standard deviations were determined.

The MW distributions of PPI and PPIH were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [41 (link)]. SDS-PAGE analysis of PPI and PPIH was performed with a 12.5% separation gel and 5% stacking gel. PPI (6 mg) and PPIH (6 mg) were separately mixed with 1 mL of buffer (0.02% bromophenol blue, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 70 mM Tris-HCl, pH 6.8). Next, the samples were heated at 95 °C for 7 min. Samples (10 μL) and protein ladders (6 μL) were loaded into separate wells. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. After staining, the gel was washed with 10% acetic acid to destain. The stained gel was digitized using an image scanner (Epson America Inc., Long Beach, CA, USA). PPIH was also analyzed by an Autoflex III mass spectrometer (Bruker Daltonik, Bremen, Germany). The masses in the range of 0–500 were measured. Each sample was analyzed in triplicate.

MALDI-ToF MS was performed on a Bruker Autoflex III Mass Spectrometer operating in positive linear mode; the analyte, matrix (DCTB) and cationisation agent (NaTFA) were dissolved in THF at a concentration of 10 mg·mL⁻¹, and then mixed in a volume ratio of 1:10:1. Then 0.3 μL of this solution was spotted onto a ground steel target plate and the solvent was allowed to evaporate prior to analysis. FlexAnalysis (Bruker) was used to analyze the data.

Small pieces of cocoons were dissolved in 8 M LiBr solution at 35 °C at a concentration of 50 μg/μL. The dissolved solutions were used for in-gel digestion of FibL followed by MALDI-TOF-MS analysis as previously reported [14 (link)]. Briefly, the LiBr solution was subjected to SDS-PAGE and the FibL band was cut out from the gel. The gel pieces were destained, washed, and digested by Trypsin Gold (Promega, Fitchburg, WI, USA). The digested peptide fragments were extracted from the gel, dried, and subjected to MALDI-TOF-MS analysis using an autoflex III mass spectrometer (Bruker Daltonics, Billerica, MA, USA).

Glutathionyl Hb was measured in cold-water hemolysates of thawed RBC concentrates by Matrix-Assisted Laser-Desorption in a Time-of-Flight mass spectrometer (MALDI-ToF) [25 (link)]. All samples were run in quadruplicate. The Hb concentration of the diluted RBCs was measured at 420 nm (EnSight™, Perkin-Elmer, Monza, Italy) and compared to that of standard human Hb (Sigma-Aldrich, St. Louis, MO, USA).
According to their individual values (20–50 µmol/L), samples were diluted to a 10 µmol/L concentration. For the MALDI analysis, a 10 µL sample was mixed to an equal volume of freshly prepared sinapinic acid matrix (Sigma-Aldrich, MALDI-grade brand, 30 mg/mL in 50% v/v acetonitrile—0.1% trifluoroacetic acid). Four one-microliter aliquots were manually spotted in adjacent circular wells of a stainless-steel plate, air-dried at room temperature, and loaded into the Bruker Autoflex III mass spectrometer for measurement as described [26 (link)]. Peak areas were measured with a custom spreadsheet, and glutathionylated-to-free thiol HbSSG/HbSH was expressed as a percent ratio.