Voyager de pro

MALDI-TOF MS was performed at the Protein Function Discovery Facility (Queen’s University, Kingston, ON, Canada) using alpha-cyano-4-hydroxycinnamic acid matrix on dried droplets with a SCIEX Voyager DE Pro in Linear mode. Protein sequencing by tandem mass spectrometry was done at the SickKids Proteomics, Analytics, Robotics & Chemical Biology Centre (SPARC, The Hospital for Sick Children, Toronto, ON, Canada).

Mass spectra were acquired with a Voyager-DE PRO (Sciex, Framingham, MA, USA) equipped with a nitrogen laser emitting at 337 nm. Ions were accelerated to a final potential of 20 kV, and the mass spectrum was the sum of 300 laser shots. An external mass calibration was used (a mixture of peptides from the Sequazyme™ standards kit, AB Sciex). The analysis was performed in linear mode (instrumental mass accuracy is 0.05%). Samples were prepared by diluting 10-fold the protein solution (0.2 mg/ml) in the matrix sinapinic acid (Sigma-Aldrich, St. Louis, MI, USA), used without further purification and dissolved in 0.1%TFA/acetonitrile (70/30 v/v). About 1 μl of the mixture was deposited onto the MALDI sample plate and let dried to complete co-crystallization.

The pGlu formation at the N-terminus of the synthetic α-synuclein79–90 peptide was monitored by mass spectrometry. 22.6 µg of this peptide were incubated in a total volume of 200 µl 50 mM Tris/HCl buffer, pH 8.0, (100 µM) for 10, 30 and 60 min in the absence or presence of the enzyme QC (0.7 µg/ml; 20 nM) with and without the QC inhibitor PBD150 (100 µM). The analytes were ionized by a nitrogen laser pulse (337 nm) and accelerated under 20 kV with a time-delayed extraction before entering the time-of-flight mass spectrometer (Voyager De Pro, Sciex). The maternal synthetic α-synuclein79–90 peptide was detected at the mass of 1130.6 Da, whereas after pGlu79 modification and liberation of ammonia the molecular weight was reduced to 1113.8 Da.

Pre-reduced recombinant hTrx1 and hGrx1 (50 µM) were incubated with 0.1 and 0.5 mM MQ, respectively, for one hour at 37 °C. Modified proteins were desalted with C4 ZipTip and then crystallized on a matrix-assisted laser desorption/ionization (MALDI) target plate with MALDI matrix prepared through dissolving 10 mg Sinapinic acid in 75% acetonitrile and 0.1% trifluoroacetic acid (TFA). MQ-modified Trx1 and Grx1 were identified using MALDI-TOF mass spectrometry (Voyager DE-Pro, AB SCIEX). Each spectrum was the result of 50 laser shots and myoglobin was used for external calibration.

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.