Oasis mcx cartridge

Five famous brands of braised chicken named H (purchased from Nanjing, Jiangsu in China), L (purchased from Suzhou, Anhui in China), K (purchased from Daokou, Henan in China), D (purchased from Dezhou, Shandong in China), and N (purchased from Nanjing, Jiangsu in China) were used as measure samples. 5,5′‐Dithiobis (2‐nitrobenzoic acid), 99% DTNB, 2,4‐dinitrophenylhydrazine (DNPH), trichloroacetic acid (TCA), thiobarbituric acid (TBA), guanidine hydrochloride, hydrochloric acid, sodium hydroxide, ethyl acetate, ethanol, methanol, ammonium hydroxide, 1,1,3,3‐Tetrathoxypropane, sodium tetraborate decahydrate, sodium borohydride, ethylenediaminetetraacetic acid disodium salt (EDTA‐Na₂), phosphate buffer saline (PBS, pH7.2–7.4), n‐hexane, and BCA kit were purchased from Nanjing Ruiyi Biological Technology Co., Ltd. The solid phase extraction (SPE) columns (Oasis MCX cartridge, 60 mg/3 ml, 30 μm) were obtained from Waters Corporation. Chicken ELISA kit was purchased from Nanjing Maibo Biotechnology Co., Ltd, China.

A total volume of 5 mL of the reaction mixture underwent extraction using 5 mL of ethyl acetate via vortexing for a duration of 5 min and ultrasonication for 10 min. This extraction process was iterated three times. Afterward, the combined ethyl acetate extraction solutions were concentrated to roughly 20 mL using nitrogen blowing. Subsequently, these solutions were introduced into a Waters Oasis MCX cartridge (3 mL, 60 mg) for elution and purification following our previously established procedure [18 (link),31 (link)].
HAs were separated, identified and quantified using a Waters Acquity UPLC system (Waters, Milford, MA, USA) equipped with a triple quadrupole mass spectrometer and Acquity UPLC BEH C18 column (50 × 2.1 mm i.d., 1.7 m) at 35 °C. The gradient elution procedure and MS parameters were the same as in our previous studies [18 (link),31 (link)].

The 20 µg of digested peptides were lyophilized and cleaned with a 1 cc Oasis MCX cartridge (Waters Corporation, Milford, MA, USA). Eluates were lyophilized and resuspended with 0.1 M triethylammonium bicarbonate, pH 8.5, to a final concentration of 1 µg/µL. A 19 µL aliquot of each sample was made, and all aliquots were then brought to 25 µL with 0.1 M TEAB, pH 8.5. TMTpro 16plex™ Isobaric Label Reagent, 0.5 mg (Thermo Scientific, Watham, MA, USA), was resuspended in 70 µL of anhydrous acetonitrile. In total, 25 µL of sample and 20 µL of TMTpro 16plex™ reagent were mixed for each channel. Labeled peptides from each experiment were multiplexed together and lyophilized. The labeled multiplexed samples for each experiment were cleaned with a 1 cc C18 Sep-Pak cartridge (Waters Corporation, Milford, MA, USA) and lyophilized.

The
PBS storage buffer was removed from the beads. To denature and reduce
the proteins bound to the beads, 30 μL of 50 mM TEAB (pH 8.5),
6 M urea, 2 M thiourea, 10 mM DTT were added and then incubated for
1 h at 35 °C. This was then followed by alkylation with 50 mM
iodoacetamide for 45 min in the dark and quenched with a final concentration
of 50 mM dithiothreitol (DTT). Samples were diluted with 50 mM TEAB
pH 8.5 to a final concentration of 1 M urea. Trypsin was then added
to a final concentration of 10 ng/μL and incubated overnight
(16 h) at 35 °C. The digested peptides were desalted with Oasis
MCX cartridge (Waters) and then dried down to ∼100 μL
using speed vacuum SC110 (Thermo Savant, Milford, MA). All samples
were filtered with 0.22 μm cellulose acetate spin filters (Costar).
Filtered peptides were then dried down to dryness in the speed vacuum.

The proteins in the DRM (fractions 2 and 3), as well as fraction 8, were extracted in 10% trichloroacetic acid on ice for 30 min and centrifuged at 20,000 g for 1h. The precipitated pellets were washed with acetone twice.
25 μg protein from the DRM and fraction 8 were reduced with DTT and alkylated by iodoacetamide treatment. Samples were treated with 2.5 μg trypsin overnight and then evaporated to dryness in a speed vacuum centrifuge. Digested samples were reconstituted in 150 μl of 100 mM TEAB. A mixture of 6 μl of 4% CH₂O and 6 μl of 0.6M NaBH₃CN was added to the sample solution for the light labeling, and a mixture of 6 μl of 4% ¹³CD₂O and 6 μl of 0.6M NaBD₃CN was added in the heavy labeling. Both solutions were incubated for 2 h. The reactions were quenched with 1% ammonia and two differentially labeled samples were acidified with formic acid (FA). They were pooled and applied to solid-phase extraction using the Oasis MCX Cartridge (Waters Corporation, Milford, MA). Samples were dissolved in 0.1% trifluoroacetic acid (TFA) and desalted with the Sep-Pak C18 Vac Cartridge (Waters Corporation).

Extraction of lyso-Gb3 and its analogues from plasma was performed by a modification of the method described in a previous report [15 (link)]. As a standard solution, charcoal-treated plasma was spiked with lyso-Gb3 at 0 to 250 nM. As an IS solution, lyso-Gb3-IS was diluted in 1% H₃PO₄/MeOH to 2.5 nM. Twenty μL aliquots of the standard solution or plasma samples from the patients, functional variants and healthy subjects were each added to 80 μL of the IS solution, followed by mixing and centrifugation for protein precipitation to remove insoluble protein. After centrifugation, an 80 μL aliquot of each supernatant was diluted in 920 μL of 1% H₃PO₄/MeOH, followed by transfer to an OASIS MCX cartridge (30mg, 60μm; Waters Corp., Milford, MA, USA) preconditioned with 1200 μL of MeOH and 1200 μL of 2% H₃PO₄. The cartridge was washed with 1200 μL of 2% FA, then with 1200 μL of 0.2% FA/MeOH, and finally with 1200 μL of 2% NH₄OH (28%[v/v])/50% MeOH. In the case of extraction of lyso-Gb3-related analogues, the final washing step was excluded. Lyso-Gb3, its analogues and lyso-Gb3-IS were eluted into glass tubes with 1200 μL of 2% NH₄OH (28%[v/v])/MeOH, followed by drying in an evaporator. Each residue was reconstituted in 30–40 μL of 0.1% FA/50% ACN. After sonication and centrifugation, the reconstituted sample was injected into the nano-LC MS/MS system.