Compass 1

The tryptic peptide samples were prepared for injection into an Ultimate3000 Nano/Capillary LC System (Thermo Scientific, Altrincham, UK) coupled to a Hybrid quadrupole Q-Tof impact II™ (Bruker Daltonics, Bremen, Germany) equipped with a Nano-captive spray ion source. Briefly, peptides were enriched on a µ-Precolumn 300 µm I.D. × 5 mm C18 Pepmap 100, 5 µm, 100 A (Thermo Scientific, UK), separated on a 75 μm I.D. × 15 cm, and packed with Acclaim PepMap RSLC C18, 2 μm, 100Å, nanoViper (Thermo Scientific, UK). Solvent A and B, containing 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile, respectively, were supplied on the analytical column. A gradient of 5–55% solvent B was used to elute the peptides at a constant flow rate of 0.30 μL/min for 30 min. Electrospray ionization was carried out at 1.6 kV using the CaptiveSpray. Mass spectra (MS) and MS/MS spectra were obtained in the positive-ion mode over the range (m/z) 150–2200 (Compass 1.9 software, Bruker Daltonics version 4.4). The LC-MS analysis of each sample was done in triplicate.

The LC-MS/MS was used to determine the quantification of the peptides from the digested samples. The tryptic peptide samples were prepared for injection into an Ultimate3000 Nano/Capillary LC System (Thermo Scientific, Gloucester, UK) coupled to a Hybrid quadrupole Q-Tof impact II™ (Bruker Daltonics, Coventry, UK) equipped with a Nano-captive spray ion source. Briefly, peptides were enriched on a µ-Precolumn 300 µm i.d. × 5 mm C18 Pepmap 100, 5 µm, 100 A (Thermo Scientific, UK), separated on a 75 μm I.D. × 15 cm and packed with Acclaim PepMap RSLC C18, 2 μm, 100Å, nanoViper (Thermo Scientific, UK). Solvent A and B containing 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile, respectively, were supplied on the analytical column. A gradient of 5% to 55% solvent B was used to elute the peptides at a constant flow rate of 0.30 μL/min for 30 min. Electrospray ionization was carried out at 1.6 kV using the CaptiveSpray. Mass spectra (MS) and MS/MS spectra were obtained in the positive-ion mode over the range (m/z) 150–2200 (Compass 1.9 software, Bruker Daltonics).

LC-MS/MS was operated on an Ultimate3000 Nano/Capillary LC System (Thermo Scientific, Waltham, MA, USA) and a Hybrid quadrupole Q-Tof impact II™ (Bruker, Billerica, MA, USA) equipped with a Nano-captive spray ion source. Briefly, 20 µL of the sample, with 1 μg of peptides, was enriched on an Acclaim™ PepMap™ 100 C18 HPLC Columns (300 µm i.d. × 5 mm length, 5 µm particle size, 100 Å pore size, Thermo Scientific, Waltham, MA, USA) equilibrated in 2% ACN and 0.1% TFA, for 8 min at 10 μL/min. with an Acclaim PepMap RSLC C18 analytical column, NanoViper (75 µm i.d. × 150 mm length, 2 µm particle size, 100 Å pore size, Thermo Scientific, Waltham, MA, USA). Mobile phase A (0.1% formic acid in water) and Mobile phase B (80% ACN containing 0.1% formic acid) were delivered to the analytical column. Peptides were eluted at 300 nL/min. by gradient the mobile phase B from 5% B to 55% for 30 min. Electrospray ionization was conducted by a CaptiveSpray nano boosted (Bruker, Billerica, MA, USA) in positive mode at 1.6 kV. Inquiry of Mass spectra (MS) and MS/MS spectra was conducted from 150 m/z to 2200 m/z (Compass 1.9 software, Bruker, Billerica, MA, USA).

The extracted peptides were analyzed with the HCTUltra LC-MS system (Bruker Daltonics Ltd; Hamburg, Germany) coupled with a nanoLC system (UltiMate 3000 LC System, Thermo Fisher Scientific; Madison, WI, USA) equipped with an electrospray. Briefly, five microliters of peptide smples separated with the flow rate of 300 nL/min on nanocolumn (Acclaim PepMap™ 100 C18 column 50 mm internal diameter 0.075 mm). Solvent A and B containing 0.1% formic acid in water and 80% acetonitrile, respectively, were used to elute peptides using a linear gradient of 4–70% of solvent B (0–20 min) followed by 90% B from 20–25 min retention time to remove all peptides in the column. Mass spectra (MS) and MS/MS spectra were obtained in the positive-ion mode over the range (m/z) 400–1500 (Compass 1.9 software, Bruker Daltonics).

Samples were introduced splitless into the ESI source. Compounds were ionized with an end plate offset of −500 V, a capillary voltage of 4,500 V in positive mode. The nebulizer gas N₂ flow was 11 Lmin-1, 3.5 bar pressure heated to 220°C. Bruker Compass 1.9 acquired the profile data with a spectra rate of 1 Hz (full scan) and a mass range from 30 to 1,300 m/z. MS/MS data were generated of the five most intense ions selected for fragmentation within a mass range of ±1–3 Da, 20–25 eV collision energy and 5-Hz spectra rate. The mass accuracy was adjusted by internal calibration using sodium format clusters in ESI+ and ESI- modes with Bruker DataAnalysis 4.4 software after calibration with Proteowizard (http://proteowizard.sourceforge.net).
For detect instrumental variations, a quality control (QC) samples was analyzed, consisting of a pool of all samples (SCZ, BD, and HC), prepared by mixing 5 µl of each sample. QC sample preparation was performed equally as for the other samples. The QC samples were injected three times at the beginning of the batch analyses, at every five injections, and once at the end of the batch.

Synthetic glycopeptides were dissolved in 50% acetonitrile (ACN) containing 0.1% formic acid (FA) and used for direct infusion experiments (500 fmol/μL) on a Q-TOF impact II (Compass 1.9, otofControl 4.0) interfaced with an electrospray ionization (ESI) Apollo source (both Bruker, Bremen, Germany). Data was acquired using a modified version of the standard Instant Expertise method in which the product-ion spectra rate was 4–16 Hz (depending upon precursor intensity) and the number of precursors selected for fragmentation is adjusted automatically to retain a MS-tandem MS duty cycle of 3 s. For this experiment, precursors were manually selected but the tandem MS spectra rate was automatically determined as above. MS spectra were acquired at 2 Hz and precursors were isolated with a width of 3–5 Da depending on m/z values. Collision energies were increased from 10 to 140 eV in steps of 10 eV; for each collision energy data was acquired for 2 min in the range 150–2300  m/z.

LC-HR-MS was performed on a Bruker maXis UHR Q-TOFmass spectrometer coupled to an Ultimate 3000 RSLC system (Dionex, Germering, Germany) with a binary pump, an autosampler, a column thermostated at 40 °C, and a DAD detector. A Dionex Acclaim RSLC 120 C18 (250 × 2.1 mm; 2.2 μm) column fitted with a C18 Security Guard Ultra (2.1 mm) (Phenomenex, Le Pecq, France) guard filter was used for LC separations. The mobile phase was water + 0.1% formic acid (solvent A) and acetonitrile + 0.08% formic acid (solvent B). A solvent gradient at a flow rate of 300 μL/min was set as follows: 3% of B for 1 min, then a linear gradient up to 70% of B during 39 min. The column was rinsed with 95% of B for 5 min and then re-equilibrated to the initial conditions for 10 min after each run. 4 μL of sample reconstituted in methanol at 10 mg/mL of engaged material were injected. UV spectra were recorded from 195 to 500 nm. The positive mode using an ESI source in the range of 50 to 1300 m/z at 1 Hz was used to acquire MS data. The parameters were as follows: capillary voltage 4.5 kV, nebulizing gas at 1.5 bar, drying gas heated at 200 °C, at 8 L/min. Data (MS and UV) were acquired with Compass 1.3 software (Bruker) and MS areas were measured with Quant Analysis 2.1 software (Bruker). Extracted ion chromatograms for DPT, PPT, PPTG, MPT and MPTG are presented in Figures S6–S10.