Hplc ms system

Cells used for the analysis of fatty acids were harvested from the third quadrants of ISP 2 agar plates. Fatty acid methyl esters (FAMEs) were extracted as described by Kuykendall, et al.^{74 (link)} and analyzed according to the instructions of the Microbial Identification System (MIDI; Microbial ID). Isoprenoid quinones were extracted using a CHCl₃/MeOH mixture (2:1, v/v) from freeze-dried cells (500 mg) and analyzed using an HPLC-MS system (Agilent)⁷⁵ . Polar lipids were extracted and separated by two-dimensional thin-layer chromatography on silica gel 60 F₂₅₄ plates (Merck). Molybdophosphoric acid, ninhydrin reagent, molybdenum blue, and α-naphthol/H₂SO₄ reagents were used for the detection of total lipids, lipids containing free aminolipids, phosphorus-containing lipids and glycolipids, respectively^{76 (link)}. The analyses of sugars and amino acids in whole cell hydrolysates were performed following previous methods⁷⁷ .

The HPLC-MS-system: Agilent HPLC-MS-system consisting of: 1200 binary pump, 1200 automatic sampler, thermostated, 1200 column thermostate, 1200 diode array detector with 10 mm standard flowcell, LC/MSD Ultra Trap System XCT 6330 (Agilent, Waldbronn).
HPLC parameters: stationary phase: Nucleosil 100 C18 3 µm, 100 × 2 mm ID with a precolumn 10 × 2 mm ID (Dr. Maisch GmbH, Ammerbuch), column temperature: 40 °C, mobile Phase: A = 0.1% formic acid, B = 0.06% formic acid in acetonitrile, gradient: t₀ = 10% B, t₁₅ = t₁₇ = 100% B, posttime 6 min. 10% B, flowrate: 400 µL/min, injection volume: 2.5 µL, detections wavelengths (bandwidth): 230 nm (10 nm), 260 nm (20 nm), 280 nm (20 nm), 360 nm (20 nm), 435 nm (40 nm), software: LC/MSD ChemStation Rev. B.01.03, Agilent.
MS parameters: ionisation: ESI (positive and negative, alternating), mode: ultra-scan, capillary voltage: 3.5 kV, temperature: 350 °C, target mass: m/z 800, software: 6300 series trap control version 6.1, Bruker Daltonik (Agilent, Waldbronn, Germany).

Biotinylated thiol
biotin-PEG₃-Cys was synthesized using Fmoc-based solid-phase
peptide synthesis.^{40 (link),41 (link)} In brief, the H-Rink amide resin
was used for the synthesis and all couplings were performed in an
orthogonal manner. Removal of the Fmoc-group was performed with 25%
piperidine in DMF for 10 min. First coupling was performed using 4
equiv of building block, 4 equiv of COMU, 4 equiv of OxymaPure, and
8 equiv of DIPEA in DMF for 30 min. Second coupling was done with
4 equiv building block, 4 equiv PyBOP, and 8 equiv DIPEA in DMF for
30 min. Residual free amines were acetylated using Ac₂O/DIPEA/DMF
(1/1/8) for 5 min. Final cleavage from resin and removal of protecting
groups was performed using four times 1 mL TFA/TIPS/ODT/H₂O (94/1/2.5/2.5). TFA was evaporated and the thiol was precipitated
by the addition of H₂O. Subsequently, the sample was flash-frozen
and lyophilized. Final purification was performed using a Nucleodur
C18 Gravity 5 μm column on an Agilent HPLC system with solvents
H₂O (with 0.1% TFA) and acetonitrile (with 0.1% TFA). Characterization
of the product was performed on an Agilent HPLC/MS system with a Nucleodur
C4 5 μm column.