Digestion was halted by addition of 0.3% v/v trifluoroacetic acid (TFA), and peptides were desalted on Waters Sep-Pak® Vac cartridges (Waters™ Cat No: WAT054955) with a wash of 1 mL 0.1% TFA followed by elution in 0.6 mL of 70% acetonitrile 0.1% formic acid (FA). Peptides were dried by speed vacuum and resuspended 50 mM triethylammonium bicarbonate. Peptide concentrations were checked by Pierce Quantitative colorimetric assay (Cat No: 23275). The same amount of peptide from each sample was then labeled for 2 hours at room temperature, with 0.5 mg of Tandem Mass Tag Pro (TMTpro) reagent (16-plex kit, manufactures instructions Thermo Fisher Scientific, TMTpro™ Isobaric Label Reagent Set; Cat No: 44520, lot no. VI310352) (18 (link)). Labelling reactions were quenched with 0.3% hydroxylamine (v/v) at room temperature for 15 min. Labeled peptides were then mixed and dried by speed vacuum. The TMT-labeled peptide mix was desalted to remove excess label using a 100 mg Waters SepPak cartridge, eluted in 70% acetonitrile, 0.1% formic acid and lyophilized to dryness.
Sep pak vac cartridge
Manufactured by Waters Corporation
Sourced in United States
Sep-Pak Vac cartridges are solid-phase extraction (SPE) devices used for sample preparation in analytical chemistry. They are designed to facilitate the isolation, purification, and concentration of analytes from complex matrices, such as biological fluids or environmental samples, prior to instrumental analysis.
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14 protocols using sep pak vac cartridge
1
TMTpro Isobaric Labeling of Peptides
2
SCFA Quantification in Mouse Stool
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To determine whether the SCFA levels are different between control, LB 1022, and OVA, stool samples were collected from each mouse at 8, 12, and 16 weeks and stored at −80°C. The samples were reconstituted in deionized water and passed through a membrane filter using Sep-Pak Vac cartridges (WAT054955; Waters, USA). Filtered samples were then analyzed using high-performance liquid chromatography (HPLC) by a refractive index detector (RefractoMAX520; ERC, Japan).
3
Peptide Purification and Tandem Mass Tagging
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We performed peptide purification and labeling according to previously published methods (Grecco et al., 2021a (link); Grecco et al., 2022a (link)). To summarize our previous methods, digestion was halted by addition of 0.3% final v/v trifluoroacetic acid (TFA), and peptides were desalted on Waters Sep-Pak® Vac cartridges (Waters™ Cat No: WAT054955) with a wash of 1 mL 0.1% TFA followed by elution in 0.6 mL of 70% acetonitrile 0.1% formic acid (FA). Peptides were dried by speed vacuum and resuspended 50 mM triethylammonium bicarbonate. Peptide concentrations were checked by Pierce Quantitative colorimetric assay (Cat No: 23275). The same amount of peptide from each sample was then labeled for 2 h at room temperature, with 0.5 mg of Tandem Mass Tag Pro (TMTpro) reagent (16-plex kit, manufactures instructions Thermo Fisher Scientific, TMTpro™ Isobaric Label Reagent Set; Cat No: 44520, lot no. VI310352) (Li et al., 2020 (link)). Labeling reactions were quenched with 0.3% hydroxylamine (final v/v) at room temperature for 15 min. Labeled peptides were then mixed and dried by speed vacuum. The TMT-labeled peptide mix was desalted to remove excess label using a 100 mg Waters SepPak cartridge, eluted in 70% acetonitrile, 0.1% formic acid and lyophilized to dryness.
4
TMTpro Isobaric Labeling of Peptides
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Digestion was halted by addition of 0.3% v/v trifluoroacetic acid (TFA), and peptides were desalted on Waters Sep-Pak® Vac cartridges (Waters™ Cat No: WAT054955) with a wash of 1 mL 0.1% TFA followed by elution in 0.6 mL of 70% acetonitrile 0.1% formic acid (FA). Peptides were dried by speed vacuum and resuspended 50 mM triethylammonium bicarbonate. Peptide concentrations were checked by Pierce Quantitative colorimetric assay (Cat No: 23275). The same amount of peptide from each sample was then labeled for 2 hours at room temperature, with 0.5 mg of Tandem Mass Tag Pro (TMTpro) reagent (16-plex kit, manufactures instructions Thermo Fisher Scientific, TMTpro™ Isobaric Label Reagent Set; Cat No: 44520, lot no. VI310352) (18 (link)). Labelling reactions were quenched with 0.3% hydroxylamine (v/v) at room temperature for 15 min. Labeled peptides were then mixed and dried by speed vacuum. The TMT-labeled peptide mix was desalted to remove excess label using a 100 mg Waters SepPak cartridge, eluted in 70% acetonitrile, 0.1% formic acid and lyophilized to dryness.
5
Quantification of Abscisic Acid and Seed Water Content
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Imbibed or germinating seeds (20 mg) were frozen in liquid N and ground using a pestle and mortar. The ground powder was immersed in 1 ml of 80% methanol and stored at 4 °C for 1 h. The extracts were filtered with C18 (Sep-Pak Vac) cartridges (Waters, USA) to remove pigments and other polar materials. The filtered solution was dried and concentrated using a rotary vacuum concentrator and suspended in Tris-buffered saline (TBS). The level of ABA was determined using the enzyme-linked immunosorbent assay (ELISA) Kit (Agdia). To measure the seed water content, ca. 330 seeds immersed in SDW were collected at a given time, and external liquid was removed using a silica-based membrane column by spinning at 12,000 rpm for 5 min. Then, dry seed weight was subtracted from the fresh seed weight to determine the amount of water absorbed by the seeds. External osmotic potential of varying concentrations of PEG4000 was calculated from the estimated osmolality (mol kg−1) (Money 1989 (link)).
6
Peptide Labeling and Quantification
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Digestions were quenched with 0.4% trifluoroacetic acid (TFA; v/v; catalog #91699, Fluka), and the resultant peptides were desalted by solid-phase extraction using Sep-Pak Vac cartridges C18 cartridges (catalog #WAT054955, Waters), lyophilized O/N, and resuspended in 55 μl of 50 mm triethylammonium bicarbonate (catalog #T7408, Sigma-Aldrich), pH 8.5. Peptides were quantified using Quantitative Colorimetric Peptide Assay (catalog #23275, Thermo Fisher Scientific) to ensure equivalent concentrations across each set of samples before being covalently labeled with TMTpro Isobaric Label Reagent 16-plex (catalog #44520, lot VI310352, Thermo Fisher Scientific) at a 1:7 peptide to TMTpro ratio. After 1 h of incubation, the labeling reaction was quenched with 0.3% hydroxylamine (v/v) for 15 min before combining the samples. The multiplexed sample was concentrated to dryness in vacuum centrifuge, reconstituted with 0.1% TFA aqueous (v/v), desalted via Waters Sep-Pak Vac cartridges as before, and lyophilized.
7
Comprehensive Lipid Analysis of Bacterial Strains
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The polar lipids of strains 172403-2 T and BT310 T were extracted (Minnikin et al. 1984 ) and examined using two-dimensional thin-layer chromatography (TLC). The separated polar lipids were identi ed by spraying several reagents as described by Komagata and Suzuki (1987) . Lipoquinones were extracted with Sep-Pak Vac cartridges (Waters) and analyzed by high-performance lipid chromatography (HPLC) method (Hiraishi et al. 1996) . For cellular fatty acids analysis, strains 172403-2 T and BT310 T were incubated on R2A agar for 3 days at 25°C. The cellular fatty acids were puri ed by saponi cation, methylation and extraction procedures as previously described (Sasser 1990 ). The fatty acid methyl esters (FAME) were identi ed using the Sherlock Microbial Identi cation System V6.01 (MIS, database TSBA6, MIDI Inc., Newark, DE, USA).
8
Fatty acid, lipid, and quinone analysis
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To analyze the composition of cellular fatty acid, polar lipid, and quinone strain BT688 T was grown on R2A agar at 25°C for 3 days and collected cells were freeze-dried. Polar lipids of strain BT688 T were extracted as described by Minnikin et al. (1984) (link). The total lipids, glycolipids, phosphatidylcholine, and amino lipid groups were separated using two-dimensional thin-layer chromatography (TLC) and detected by using proper detection reagents (Komagata and Suzuki 1987) (link). Fatty acids were puri ed by saponi cation, methylation, and extraction procedures (Sasser 1990 ). Lipoquinones were extracted using the Sep-Pak Vac cartridges (Waters) and analyzed by high-performance lipid chromatography (HPLC) based on previous methods (Hiraishi et al. 1996) (link). The fatty acid methyl esters (FAME) were identi ed using the Sherlock Microbial Identi cation System V6.01 (MIS, database TSBA6, MIDI Inc).
9
Lipid Composition Analysis of Bacterial Strains
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To analyze the cellular composition of polar lipid, fatty acid, and quinone of strains BT290 T and BT689 T , both strains were grown on R2A agar at 25°C for three days and then cells were freeze-dried. Polar lipids were extracted as described previously (Minnikin et al. 1984) . Total lipids, glycolipids, phosphatidylcholine, and amino groups were separated using two-dimensional thin-layer chromatography (TLC). The polar lipid spots were detected by spraying the proper detection reagents (Komagata et al. 1987; Minnikin et al. 1984 ). The fatty acids were puri ed by saponi cation, methylation, and extraction procedures and analyzed by Sherlock Microbial Identi cation System V6.01 (MIS, database TSBA6, MIDI Inc., Newark, DE, USA) (Sasser et al. 1990 ). The quinones of strains BT290 T and BT689 T were extracted using the Sep-Pak Vac cartridges (Waters) and analyzed by high-performance lipid chromatography (HPLC) based on the previous methods (Hiraishi et al. 1996) . The fatty acid methyl esters (FAME) were identi ed using the Sherlock Microbial Identi cation System V6.01 (MIS, database TSBA6, MIDI Inc).
10
Lipid Profiling of Bacterial Strain BT186
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To analyze the polar lipid, fatty acid, and lipoquinone components of strain BT186 T , cells were grown on R2A agar at 25 °C for three days. After then, cells were harvested and freeze-dried. The total lipids, glycolipids, phosphatidylcholine, and amino groups were separated using two-dimensional thin-layer chromatography (TLC). The polar lipid spots were detected by spraying the proper detection reagents as previously described (Komagata and Suzuki 1987; Minnikin et al. 1984) (link). The cellular fatty acids methyl esters (FAME) of strain BT186 T were analyzed using the Sherlock Microbial Identi cation System V6.01 (MIS, database TSBA6, MIDI Inc) according to the protocol described by Sasser (1990) . The respiratory quinone was extracted using the Sep-Pak Vac cartridges (Waters) and analyzed by high-performance lipid chromatography (HPLC) follow the previous methods (Hiraishi et al. 1996) (link).
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