Hlb column

On-bead digestion in preparation for LC-MS/MS was performed following an established protocol.[49 (link)] Digestion buffer (50 mM NH₄HCO₃) was added to the beads, and the mixture was treated with 1 mM dithiothreitol (DTT) at room temperature for 30 minutes, followed by 5 mM iodoacetimide (IAA) at room temperature for 30 minutes in the dark. Proteins were then digested overnight with 2 μg of lysyl endopeptidase (Wako) at room temperature and further digested overnight with 2 μg trypsin (Promega) at room temperature. Resulting peptides were desalted with HLB column (Waters) and were dried under vacuum.

Each tissue sample was added to 250 μL of urea lysis buffer (8 M urea, 10 mM Tris, 100 mM NaH2PO4, pH 8.5, including 2.5 μL (100x stock) HALT(-EDTA) protease and phosphatase inhibitor cocktail (Pierce)) in a 1.5 mL Rino tube (Next Advance) harboring stainless steel beads (0.9–2 mm in diameter). Samples were homogenized twice for 5-minute intervals in the cold room (4 °C). Protein homogenates were transferred to 1.5 mL Eppendorf tubes on ice and were sonicated (Sonic Dismembrator, Fisher Scientific) thrice for 5 sec each with 5 sec intervals of rest at 30% amplitude to disrupt nucleic acids and were subsequently centrifuged at 4° C. Protein concentration was determined by the bicinchoninic acid (BCA) method, and samples were frozen in aliquots at −80 °C. Protein homogenates (100 μg) were treated with 1 mM dithiothreitol (DTT) at room temperature for 30 min, followed by 5 mM iodoacetimide at room temperature for 30 min in the dark. Protein samples were digested with 1:25 (w/w) lysyl endopeptidase (Wako) at room temperature overnight. Next day, samples were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and were further digested overnight with 1:25 (w/w) trypsin (Promega) at room temperature. The resulting peptides were desalted with HLB column (Waters) and were dried under vacuum.

The pooled samples were fractionated by isoelectric focusing with an Off-Gel Fractionator (OG) (Agilent Technologies, California, USA) and 24-well IPG strips (with a nonlinear gradient from pH 3 to pH 10) according to the manufacturer’s protocol. After fractionation, each of the 24 fractions was desalted again with an HLB column (Waters, Massachusetts, USA) prior to nanoLC-Orbitrap MS/MS analysis.

Approximately 0.1–0.2 g of tissue from selected stages of S.E. were sampled and frozen at −70 °C until further analysis. The measurement of endogenous free IAA and IAA metabolites were performed as previously described (40 ), with some modifications. Approximately 200 mg of each sample were ground to a fine powder in liquid nitrogen, followed by extraction with 800 μl precooled 80% methanol solution containing 1% acetic acid, vigorous shaking in the dark overnight at 4 °C, and centrifugation at 15,871 g at 4 °C for 15 min. The supernatant was carefully collected, and the pellet was resuspended with additional 0.4 ml extraction buffer, extracted for 4 h at 4 °C, followed by centrifugation. The supernatants were combined and loaded onto an HLB column (Waters) and washed with 70% methanol solution containing 2% acetic acid. The filtrates were dried through evaporation under the flow of nitrogen gas and dissolved in 60 μl 10% methanol.

For pre-treatment, urine samples (10 mL) were thawing and centrifuging at 13 000 rpm for 10 min (4 °C), and 2 mL supernatant was loaded on a pre-activity HLB column (6 cm³, 200 mg, Waters Oasis, Ireland) directly. Then, it was orderly eluted by 6 mL of 5% methanol and 6 mL of methanol. The methanol eluate was collected and dried under nitrogen gas at room temperature. The residue was reconstituted in 300 μL 60% methanol–water (v/v).