Nano LC-Orbitrap-MS/MS

Separation of peptides was performed on an Ultimate 3000RSLC nano system equipped with an RP-C18 guided column (2 μm, 2 cm×100 μm,) and an RP-C18 analytical column (2 μm, 150 mm×75 μm).Ultrapure water and acetonitrile containing 0.1% formic acid (FA) was used as solvent A and B, respectively, at a flow rate of 300 nL/min. The linear gradient program was: 0 min, 4% B; 6 min, 4% B; 50 min, 25% B; 58 min, 40% B；61 min, 85% B. The column was washed with 85% B for 5 min and re-equilibrated with 4% B for 7 min before the injection of next sample. The injection volume was 2.0 μL.

The OrbitrapFusion mass spectrometer equipped with electron transfer dissociation (ETD) fragmentation mode was employed to the mapping of proteolytic peptides. The analytical parameters were: positive mode, mass range of 350-1800 m/z, MS resolution of 60,000, MS/MS resolution of 15,000, normalized collision energy of 30 eV.

References：

Zhang L, Xu L, Tu ZC, Wang HH, Luo J, Ma TX. Mechanisms of isoquercitrin attenuates ovalbumin glycation: Investigation by spectroscopy, spectrometry and molecular docking. Food Chem. 2020 Mar 30;309:125667. doi: 10.1016/j.foodchem.2019.125667. Epub 2019 Oct 19. PMID: 31679851.

UHPLC

Chromatographic separation of SPLFA and SPLF was done on an Agilent 1290 UHPLC system coupled to an auto sampler, a diode array detector (DAD) and an ACQUITY UHPLC? HSS T3 column (2.1 ×100 mm, 1.8 μm; Waters, Milford, MA, USA)maintained at 30 ℃. Eluent A was 0.05% formic acid in water and eluent B was 0.05% formic acid in acetonitrile. Elution of the SPLPA compounds was achieved using the following linear gradient elution (in %B): 0 min, 5%; 20 min, 60%; 20.1 min, 5%; 30 min, 5%, and for the SPLF compounds: 0 min, 20%, 25 min, 50%, 25.1 min, 20%, 35 min, 20%. The injection volume was 5 μL, with a flow rate of 0.3 mL/ min. All sample solutions were filtered through a 0.22 μm PTFE filter (Agilent Technologies) before analysis. The detection wavelength was 320 nm for SPLPA and 254 nm for SPLF.

References：

[1] Luo D , Mu T , Sun H .Profiling of phenolic acids and flavonoids in sweet potato (Ipomoea batatas L.) leaves and evaluation of their anti-oxidant and hypoglycemic activities[J].Food Bioscience, 2020, 39(44):100801.DOI:10.1016/j.fbio.2020.100801.

HPLC-DAD

The identification and quantification of phenolics in the S. miltiorrhiza leaves extract before and after enrichment were analyzed using an Agilent 1260 Infinity LC system (Agilent Co., Palo Alto, CA, USA).10 μL of sample dissolved in 80 % methanol was injected into the HPLC system, and phenolics were chromatographed over a YMC-Pack ODS-A (250 × 4.6 mm, 5 μm) column at room temperature with a combination of 0.1 % trifluoroacetic acid (A) and acetonitrile (B) at a flow rate of 1 mL/min. The procedure for elution was as follows: 0? 5 min, 10 % B; 5? 10 min, 10 %–12 % B; 10? 20 min, 12 %–15 % B; 20? 25 min, 15 %–20 % B; 25? 60 min, 20 %–30 % B; 60? 65 min, 30 %–100 % B; 65? 70 min, 100 % B; 70? 80 min, 100 %-10 % B. Phenolics were detected by a diode array detector (DAD) at 280 nm.

References：

[1] Hou M , Hu W , Hao K ,et al.Enhancing the potential exploitation of Salvia miltiorrhiza Bunge: Extraction, enrichment and HPLC-DAD analysis of bioactive phenolics from its leaves[J].Industrial Crops and Products, 2020, 158:113019.DOI:10.1016/j.indcrop.2020.113019.