Esi calibration mixture

The samples after CIEX chromatography were S-alkylated with iodacetamide and digested in-solution with trypsin (Promega, WI, USA). The digested samples were loaded on an ACQUITY PRM HSST 3.1 C18 column (1.8 µm, 2.1 × 150 mm, Waters, MA, USA). 0.1% formic acid was used as aqueous solvent (A) and a mixture of 80% Acetonitrile and 20% A was used as elution buffer (B). A gradient from 3.5% B to 40% B in 35 min was applied and followed by a gradient from 40% B to 95% B in 5 min at a flow rate of 6 µL/min. The second gradient should facilitate the elution of large peptides. An Agilent Series 6560 LC-IMS-Q-TOF–MS instrument (Agilent, CA, USA) with the Jetstream ESI source in positive ion, switching to MS–MS mode for eluting peaks (DDA mode) was used for detection. MS-scans were recorded from 400 – 3200 Da and the 5 highest peaks were selected for fragmentation. Calibration was done using ESI calibration mixture (Agilent, CA, USA).
Norleucine was quantified by using the software Skyline (version 21.2) and integration of the extracted-ion chromatograms of the first four isotopic peaks of the peptides. Additionally, the analysis files were converted to mgf files for further analysis using MS/MS ion search with X!-Tandem. The files were searched against an E. coli database, additionally containing the sequence of the used Fab FTN2.

Relevant protein bands were cut out and digested in gel. S-alkylation with iodoacetamide and digestion with sequencing grade modified trypsin (Promega) were performed. The peptide mixture was analysed using a Dionex Ultimate 3000 system directly linked to a QTOF instrument (maXis 4G ETD, Bruker) equipped with the standard ESI source in the positive ion, DDA mode (= switching to MSMS mode for eluting peaks). MS-scans were recorded (range: 150–2200 Da) and the 6 highest peaks were selected for fragmentation. Instrument calibration was performed using ESIcalibration mixture (Agilent). For separation of the peptides a Thermo BioBasic C18 separation column (5 μm particle size, 150°0.360 mm) was used. A gradient from 95% solvent A and 5% solvent B (Solvent A: 65 mM ammonium formiate buffer, B: 100% ACCN) to 32% B in 45 min was applied, followed by a 15 min gradient from 32% B to 75% B, at a flow rate of 6 μL∙min⁻¹. The analysis files were converted using Data Analysis 4.0 (Bruker) to XML files, which are suitable to perform MS/MS ion searches with MASCOT (embedded in ProteinScape 3.0, Bruker) for protein identification. Only proteins identified with at least 2 peptides with a protein score higher than 80 were accepted. For searches the SwissProt database was used.

All samples were measured in positive mode with a quadrupole time‐of‐flight (Q‐TOF) instrument (maXis 4G; Bruker). Standard source settings (capillary voltage 4.5 kV, nebulizer gas pressure 0.5 bar, drying gas 5 L/min, 200°C) were used. For a run, purified samples were loaded on a PGC column (100 mm x 0.32 mm, 5 μm; Thermo Fisher Scientific) using 80 mM ammonium formate buffer of pH 3.0 as aqueous solvent. A linear gradient from 1% solvent B (80% acetonitrile plus 20% solvent A) to 65% solvent B in 39 min was applied, at a flow rate of 6 μL/min. Detection was performed with the Q‐TOF instrument equipped with a standard ESI source in data‐dependent acquisition mode (switching to MS/MS mode for eluted peaks) that is directly linked to the Thermo Ultimate 3000 UPLC system. MS scans were recorded within a range of 150 to 2200 Da. Instrument calibration was performed with an ESI calibration mixture (Agilent).
Initial data processing was done with DataAnalysis 4.0 and QuantAnalysis 2.0 (Bruker). Known differences in the retention time during PGC‐separation identified the type of sialylgalactose linkage [22 (link)]. Due to inherent ambiguity with N‐glycans harboring three or more antennae, linkage positions were only determined for biantennary structures.

The OMV preparations from three independent biological replicates were separated in 10‐μl aliquots, each, by SDS–PAGE. Three zones were excised per lane and proteins were S‐alkylated with iodoacetamide and subsequently subjected to in‐gel digestion with modified trypsin (Promega, Mannheim, Germany). The peptide mixture was analyzed in the positive‐ion DDA mode (switching to MSMS mode for eluting peaks) using a Dionex Ultimate 3000 system (Thermo Scientific, Vienna, Austria) directly linked to a quadrupole time‐of‐flight mass spectrometer (MS) (Bruker maxis 4G ETD; Bruker) equipped with the captive spray source and nano‐booster set‐up. MS scans were recorded over a range of 150–2200 Da and the eight highest peaks were selected for fragmentation. Instrument calibration was performed using the ESI calibration mixture (Agilent, Santa Clara, CA). For separation of the peptides, a Thermo Acclaim PepMap300 RSLC C18 separation column (2‐μm particle size; 150 × 0.075 mm) was used with a Thermo Acclaim PepMap μ‐pre‐column. A gradient from 5% to 32% of solvent B (0.1% formic acid in acetonitrile) in solvent A (0.1% formic acid in HQ‐water) within 60 min was applied at a flow rate of 0.3 μl min⁻¹, followed by a 10‐min gradient from 32% B to 70% B to promote elution of large peptides.

The samples were deglycosylated using PNGase F prior to the measurement. To determine the correct pairing of the heterodimer chains, the proteins were directly injected to a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) system (LC: Dionex Ultimate 3000 LC). A gradient from 20 to 80% acetonitrile in 0.05% trifluoroacetic acid (using a Thermo ProSwift™ RP-4H column of 0.2 x 250 mm dimensions) at a flow rate of 8 μL/min was applied (30 min gradient time). Detection was performed with a Q-TOF instrument (Bruker maXis 4G) equipped with the standard ESI source in positive ion, MS mode (range: 750–5000 Da). Instrument calibration was performed using ESI calibration mixture (Agilent). Data was processed using Data Analysis 4.0 (Bruker) and the spectrum was deconvoluted by MaxEnt.

Twenty microliter PNGase F digested sample (β = 0.30 mg·mL⁻¹) was analyzed using a Dionex Ultimate 3000 system directly linked to a QTOF instrument (maXis 4G ETD; Bruker, Billerica, MA, USA) equipped with the standard ESI source in the positive ion mode. MS scans were recorded within a range from 400 to 3800 m/z. Instrument calibration was performed using ESI calibration mixture (Agilent). For separation of the proteins, a Thermo ProSwift™ RP‐4H Analytical separation column (250 × 0.200 mm) was used. A gradient from 80% solvent A and 20% solvent B (A: 0.05% trifluoroacetic acid, B: 80% acetonitrile, and 20% A) to 65% B in 20 min was applied, followed by a 15‐min gradient from 65% B to 95% B, at a flow rate of 8 μL·min⁻¹ and at 60 °C. Deconvolution of summed spectra was done using the MaxEnt algorithm in Data Analysis 4.0.