Peptides were fractionated according to their pI using the Agilent OFFGEL 3100 fractionator (Agilent Technologies). Urea 1M was added to the IEF buffer instead of 5% glycerol mentioned in the manual. Commercially available IPG dry strips 24 cm, with a linear pH gradient ranging from 3–10 (GE-Healthcare) were used. The strips were rehydrated with 40 μl/well rehydration solution in the assembled device for 30 min. 150 μl of the pooled SCX fractions redissolved in water were loaded on each well. The cover fluid (mineral oil, Agilent Technologies) was added to both ends of the gel strip. The focusing method OG24PE01, as supplied by the manufacturer was used in the first OFFGEL experiment (Experiment 1). Typical voltage ranging from 500 to 4500 V was applied until 50 kVh was reached. The maximum current was set to 50 mA and the maximum power to 200 mW. In the subsequent two biological independent samples a longer focusing was applied till 80 kVh (Experiments 2 and 3). The higher volt-hour improved separation. In total 24 fractions were collected per OFFGEL experiment. Each OFFGEL fraction was pre-cleaned using the HLB μElution plates (Waters). The eluates were dried in a SpeedVac, redissolved in 20 μl 0.1% TFA, and subjected to LC-MS/MS analysis.
Hlb μelution plate
Manufactured by Waters Corporation
Sourced in United States
The HLB μElution plate is a solid-phase extraction (SPE) microplate designed for the efficient extraction and concentration of analytes from various sample matrices. It utilizes a hydrophilic-lipophilic balanced (HLB) sorbent to provide broad-spectrum retention of both polar and non-polar compounds. The compact, 96-well format allows for parallel processing of multiple samples, enabling high-throughput sample preparation.
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6 protocols using hlb μelution plate
1
Peptide Fractionation and Analysis
2
Quantitative CXCL12 Proteoform Analysis
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The eluates were thawed for 20–30 min at room temperature and 55 μL samples, calibration standards (CALs), QC samples or blank were transferred to a Protein LoBind 96 well plate (0030 504.100, Eppendorf, Hamburg, Germany). To denature the samples, 79 μL denaturing buffer was added, resulting in a concentration of approximately 3.4 M urea, 5 mM TCEP, and 1 μg carrier protein (E. coli lysate), and a heavy isotopologue of the CXCL121–67 proteoform (ThermoFisher Scientific) was used as an internal standard. Samples were incubated for 30 min at 37°C before alkylating the cysteines with 10 mM iodoacetamide for 45 min at 25°C in the dark. Samples were split in two and digested with 5 ng/μL trypsin or 2.5 ng/μL GluC overnight at 37°C. Digestion was stopped by acidifying with TFA to a pH of 2–3. Then labeled peptides (ThermoFisher Scientific) were added as additional internal standards, and a solid‐phase extraction was performed on HLB μElution plates (18600182BA, Waters). Desalted peptides were resuspended in 2% ACN, 0.05% TFA in water containing iRT peptides (Ki‐3002‐2, Biognosys, Schlieren, Switzerland) before liquid chromatography mass spectrometry measurements.
3
Peptide Desalting and Purification
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Samples were transferred to an OASIS microplate (Waters HLB μElution plate, 186001828BA) for desalting. After binding peptides to the columns, they were washed two times with 0.05% formic acid and finally eluted with 0.05% formic acid/80% acetonitrile. Peptides were dried.
4
Metabolite Extraction Using Mixer Mill
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The samples were extracted with 50 μL of 80% MeOH per mg dry weight using a mixer mill (MM300, Retsch) with zirconia beads for 10 min at 20 Hz and 4°C. After centrifugation for 10 min, the supernatant was filtered using an HLB μElution plate (Waters).
5
Withanolides Isolation and Profiling
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The authentic samples of withanolides were isolated from plants as described in our previous paper (Ozawa et al., 2013 (link)). The leaves of P. alkekengi and P. peruviana were collected at five different developmental stages of the growing season before bearing fruit, because our pilot study showed to detect withanolides of P. alkekengi and P. peruviana at these stages (data not shown). Fresh samples of leaves at five different developmental stages were extracted with 5 μl of 80% MeOH containing 2.5 μM lidocaine (internal standard) per mg fresh weight using a mixer mill with zirconia beads (7 min at 18 Hz and 4°C). After 10-min centrifugation, the supernatant was filtered using an HLB μElution plate (Waters). The extracts (1 μl) were analyzed with LC-QTOF-MS (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). The analytical conditions for metabolite profiling were as described in elsewhere (Tamura et al., 2014 (link)). The polarity of electrospray ionization was applied in positive ionization mode.
6
Tetranor-PGDM Quantification in Artificial Urine
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Artificial urine (Supplementary Table 1 ) was spiked with tetranor-PGDM in distilled water at four levels (1.25, 2.5, 5.0, and 10.0 ng/mL). For solid-phase extraction (SPE), 0.4 mL of the urine sample was diluted to 0.8 mL with 0.1% (v/v) formic acid. The mixed solutions were applied to an SPE cartridge (HLB μElution plate, Waters, Massachusetts, USA) preconditioned with 200 μL acetonitrile and distilled water. After washing with 200 μL distilled water and 200 μL hexane, the lipid fractions were eluted with 50 μL acetonitrile. The eluate was collected and dried in vacuo. The resulting residue was reconstituted in 0.4 mL of assay solution. The sample solution containing tetranor-PGDM was then introduced into the EIA. At the same time, tetranor-PGDM was measured by LC-MS/MS as previously described [2 (link), 3 (link)]. The recovery from the SPE procedure was found to be 77.1% by LC-MS/MS (Supplementary Table 2 ).
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