Impact hd

MS² analyses were performed using high-resolution Q-TOF mass spectrometry (Bruker impactHD) coupled with an ESI source with the parameters as followed: positive-ion mode, capillary source voltage at 3500 V, drying-gas flow rate at 4 L/min, drying-gas temperature at 200 °C, and end plate offset voltage at 500 V. MS full scan mode was operated from m/z 50–1500 (100 ms scan time) with a resolution of 40,000 at m/z 1222.

¹H and ¹³C NMR spectra were recorded on Bruker Ultrashield 300 MHz or Varian
VNMR-S 400 MHz instruments. Chemical shifts (δ) are reported
in ppm relative to SiMe₄. HR-MS spectra were obtained with
a Bruker Impact HD, QTOF MS spectrometer. UV and ECD spectra were
recorded in spectroscopic grade cyclohexane or acetonitrile using
a JASCO J-810 instrument. The UV and ECD measurements were performed
in quartz cell (0.5 mm path length), at a scanning speed of −50
nm min^–1 and a resolution of 0.5 nm. The concentrations
of the samples are collected in Table SI_1. FT-IR spectra were measured on a Nicolet iS 50 spectrometer using
ATR module. A JASCO P-2000 polarimeter was used for optical rotation
([α]_D) measurements (carried out at ca. 20 °C).
Column chromatography was performed on J. T. Baker Silica Gel 40 μm
(chromatography grade). Merck Kieselgel type 60F₂₅₄ analytical
plates were used for TLC analyses. Melting points were measured on
Büchi Melting Point B-545 and uncorrected. All reagents were
used as purchased from commercial suppliers. All solvents were provided
by local suppliers and were purified by conventional methods prior
to use.
(R)-Methyl 2-cyclohexyl-2-hydroxyacetate
was prepared according to the literature procedure.^22h

To determine the product of the CsiD-catalyzed oxidative reaction using 2-KG as the substate, the reaction solution containing 10 mM 2-KG, 2 mM ascorbate, 0.25 mM Fe²⁺, 0.25 mg mL⁻¹ purified CsiD in 50 mM Tris-HCl (pH 7.4) was incubated aerobically at 30 °C and 180 rpm for 1 h. For the product of CsiD-catalyzed glutarate hydroxylation, 10 mM glutarate was added besides the above components. The product of LhgO catalyzed dehydrogenation of l-2-HG was investigated in the reaction solutions containing 50 mM Tris-HCl (pH 7.4), 5 mM l-2-HG, 0.11 mg mL⁻¹ purified LhgO, and 1 mM MTT at 30 °C and 180 rpm for 1 h. The mixture was boiled to terminate the reaction, centrifuged at 20,000 × g for 15 min, and then subjected to HPLC analysis using an Aminex HPX-87H column (Bio-Rad) and a refractive index detector^{59 (link)}. The reaction with denatured protein was conducted under identical conditions as a control. The catalytic product of CsiD was also analyzed by liquid chromatography-tandem mass spectrometry (LC–MS/MS, impact HD; Bruker Daltonics) using 0.1% formic acid at a flow rate of 0.4 mL min⁻¹ with a HPLC system coupled by negative electrospray ionization.

N-glycans were enzymatically liberated from antibodies and labeled with 2-aminobenzamide (2-AB) for fluorescence detection and quantification employing hydrophilic interaction liquid chromatography (HILIC). Structure identification was performed based on mass spectrometry (MS). Antibodies were denatured and reduced prior to enzymatic release of N-glycans using PNGase F within polyacrylamide gel blocks. Liberated glycans were fluorescently labeled using the LudgerTag 2-AB (2-aminobenzamide) Glycan Labeling Kit (Ludger, Oxford, UK). Labeled glycans were separated with an ACQUITY UPLC Glycan BEH Amide Column (Waters, Milford, MA, USA). Glycan structures of each peak were identified by MS. Therefore, either MALDI-TOF MS/post source dissociation MS (Microflex, Bruker; Billerica, MA, USA) of fractionated N-glycans co-crystallized with dihydroxybenzoic acid or online LC-ESI-qTOF CID-MS/MS (Impact HD, Bruker) was used. Glycan structures of each peak were then analyzed using a combination of MS and MS/MS fragmentation data with the Compass DataAnalysis software 4.3 (Bruker). Quantification was based on fluorescence signals using the Empower 3 software (Waters).

Antibodies were denatured by RapiGest™ SF (Waters Inc., #186002123) and tris-(2-carboxyethyl)phosphine (120 min, 95°C). N-acetylglucosamine-linked oligosaccharides were released by enzymatic digestion with Rapid PNGase F (10 min, 55°C) (Waters Inc., #186007990) followed by fluorescence tagging with RapiFluor-MS reagent (Waters Inc., #186007989) in dimethylformamide for 5 min at room temperature (RT). For clean-up of tagged glycans a μElution Plate (HILIC SPE, Waters Inc., #186002780) was used. Labeled N-glycans were analyzed by LC–MS employing a HILIC phase (Acquity UPLC BEH GLYCAN 1.7 μ, 2.1 × 150 mm; Waters Inc., #186004742) with an I-class UPLC (Waters Inc.) coupled to a high resolution QTOF mass spectrometer (Impact HD; Bruker Daltonik). Labeled N-glycans are separated using a gradient from 22% B to 44% B within 82 min (mobile phase A: acetonitrile; mobile phase B: 100 mM ammonium formate in H2O, pH4.4). RapiFluor-MS tagged N-glycans were detected with a fluorescence detector at 265 nm excitation wavelength and 425 nm emission wavelength. Fluorescence signals were employed for glycan quantification. Identification of glycan structures was performed by MS and a series of MS/MS experiments using DataAnalysis Software 4.4 (Bruker Daltonik).

The product was identified by using the HPLC analysis, specific optical rotations, and mass spectrometry. The high performance liquid chromatography (HPLC) analysis method was referred to in “Crude ADH preparation, ADH purification, and enzyme assay.” Specific optical rotations were determined by using the polarimeter (INESA WZZ-3, China). Mass spectrum (BRUKER impactHD, Germany) was performed in the negative ion detection mode with the ESI ion source.