Pal autosampler

Lyophilized peptides were reconstituted in 0.1% FA with 2% ACN containing 0.01% DDM and injected using a PAL autosampler (CTC Analytics AG, Switzerland). The sample was concentrated into an online SPE column (150 μm i.d., 360 μm o.d., 4 cm long) and separated using a 50 μm i.d., 360 μm o.d., 50 cm long column packed with 3-μm C18 particles (300-Å pore size; Phenomenex, Terrence, CA). The nanoLC separation used a Dionex UltiMate NCP-3200RS system (Thermo Scientific, Waltham, MA) with mobile phases of water with 0.1% FA (buffer A) and ACN with 0.1% FA (buffer B). Peptides were separated through a linear gradient from 8% to 35% buffer B over 100 min at a flow rate of 150 nL/min. The separated peptides were analyzed using a Thermo Scientific Q Exactive Plus for method optimization with iBASIL. Data were acquired in a data-dependent mode with MS scans from m/z 300–1800 at a resolution of 35,000 at m/z 400. Top 10 precursor ions were selected for MS/MS sequencing at a higher-energy collision dissociation (HCD) energy of 35%. The MS/MS scan resolution was set at 70,000. Different AGC settings and maximum ITs at MS/MS level were tested for optimization (see supplemental Table S1).

To separate peptides, off-line ERLIC or ERLIC-based separation was performed (14 (link)). The following conditions were adapted and optimized to obtain the highest number of identified proteins from P2′ and SV samples. Mobile phase solvent preparation was as follows: Solvents were freshly prepared for each experiment using liquid chromatography/mass spectrometry grade acetonitrile (ACN), formic acid (FA), and water from Thermo Fisher Chemicals. Solvent A was prepared as follows: 90% ACN, 0.1% FA. Ammonium hydroxide (NH₄OH, 25% weight/weight [wt/wt] in water; Fluka) was then added to adjust pH at 4.5. Solvent B was prepared as follows: 30% ACN, 0.1% FA. The digested peptide mixture was resuspended with 20 µL of solvent A and injected into a weak anion exchange PolyWax column (1-mm inner diameter × 150 mm, 5-mm particle size, 300-Å pore size; PolyLC Inc.) using a PAL autosampler (CTC Analytics) for automatic injection and fractions collection, using a gradient mode (3 min solvent A, to 10% B in 7 min, 10% B to 25% B in 24 min, 25% B to 70% B in 16 min, 70% B to 81% B in 6 min, 81% B to 100% B in 3 min, with final wash at 100% B for 6 min and reequilibration at 100% A for 20 min) at a flow rate of 40 μL/min. Twenty-four fractions were collected every 3 min between 0 and 72 min and subsequently concentrated to dryness using a speed vacuum Genevac EZ-2 Elite (SP Scientific).

The YPD medium at different stages during fermentation 2 was analyzed by means of GC-MS in order to demonstrate the matrix complexity of the fermentation samples. A sample volume of 2 μl was injected by a PAL auto-sampler (CTC Analytics, Zwingen, Switzerland) into the QP5050 (Shimadzu, Kyoto, Japan) GC-MS system. Between measurements, the autosampler was washed with DI water for 3 min. The injection temperature was set at 200°C (split ratio 1:100). For the separation, a ZB-WAX-plus column (Torrance, CA, United States, 30 m × 0.25 mm; film thickness 0.25 μm) was utilized at the following temperature program: started at 50°C for 1 min, then raised to 200°C at 20 K/min and held at the final temperature 250°C for 10 min. The transfer line to the mass spectrometer and the source temperatures were 220 and 200°C, respectively. The ionization of the compounds was performed at an acceleration voltage of 70 eV. Mass spectra were recorded in TIC mode at the m/z range of 40–600. All samples were measured in triplicate.

Discovery experiments were performed on an hybrid quadrupole-orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a ESI ion source coupled to a Surveyor HPLC-MS pump (Thermo Fisher Scientific, San Jose, CA, USA) and a PAL Auto-sampler (CTC Analytics, Switzerland).
Quantitative analyses were performed on an 4000 QTRAP mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with a Turbo V ion source coupled to an Agilent 1290 series high pressure liquid chromatography (Agilent technologies, Waldbronn, Germany).

The stable isotope abundances of water samples were analysed at the isotope facility of the IZW Berlin, using a Piccaro L1102-i water analyser (Piccaro, Santa Clara, CA, USA). Water samples were introduced into the vaporization chamber with the injection port using an attached PAL autosampler (CTC Analytics AG, Zwingen, Switzerland). Approximately 1 μl of water sample was injected into a heated vaporizer (140 °C) and then transferred to the cavity of the spectroscopic analyser where isotopologue concentrations were determined by cavity ring-down spectroscopy. Three international reference materials (SLAP, GISP and VSMOW) and an additional in-house reference material (calibrated against VSMOW-SLAP scale) were included in each batch to correct the raw values via three-point regression line (SLAP, GISP and VSMOW). The results are expressed in delta per mil notation (δ, ‰) relative to the international standard VSMOW. Precision of the measurements was better than 1.4 ‰ for δ²H and 0.3 ‰ for δ¹⁸O. The d-excess (d = δ²H − 8δ¹⁸O, [1 (link)]) was calculated for all samples.
We used R (version 3.1.1, R development Core Team, 2014 [27 ]) for calculation of the linear regression lines and respective 95 % confidence intervals (CIs), and excel spreadsheets for calculations of d-excess.