Tripletof 5600 plus mass spectrometer

The anti-counterfeiting validation experiment was performed using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-Q-TOF–MS/MS), an analytic technology inferior to UHPLC-Q-orbitrap MS/MS. The chromatography analysis protocol was based on our previous studies [25 (link), 26 (link)]; while the MS spectra monitoring was achieved using a Q − TOF − MS/MS apparatus (i.e., Triple TOF 5600^plus mass spectrometer, AB SCIEX, Framingham, MA, U.S.A.) [27 (link)]. However, the analytes were 6 counterfeit Wushicha Granules, i.e., CWG 1–CWG 6. For comparison, the Wushicha sample solution was also prepared for this analysis, under the same conditions.

We used an established LC-MS/MS-based data-independent acquisition (DIA) proteomics approach [23 (link),24 (link)] to quantify the relative abundance of serine hydrolases enriched by ActivX™ desthiobiotin-FP probe from HlungS9 in the ABPP experiment. The analysis was carried out on a TripleTOF 5600 plus mass spectrometer (AB Sciex, Framingham, MA, USA) coupled with an Eksigent 2D plus LC system (Eksigent Technologies, Dublin, CA, USA). Briefly, we used a trapping column (ChromXP C18-CL, 120 Å, 5 mm, 0.3 mm cartridge; Eksigent Technologies) to load the samples and an analytical column (ChromXP C18-CL, 120 Å, 150 × 0.3 mm², 5 mm; Eksigent Technologies) to separate peptides. The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile (ACN) containing 0.1% formic acid (B). Mobile phase A was delivered at a flow rate of 10 μL/min for 3 min to load 1~2 µg proteins to the trapping column. A gradient elution at a flow rate of 5 μL/min was used to separate the injected peptides on the analytical column. The gradient parameters are summarized in Table S2. A blank sample (30% ACN, v/v) was injected between each run to minimize carryover. MS data were collected in a positive mode with the source temperature set at 280 °C and the ion spray voltage of 3000 V for ionization.

The polypeptide samples were diluted to 1 μg/μL using the buffer, the sample volume was set to 5 μL and the scanning mode was collected for 60 min. Peptides with mass to charge ratios of 350–1200 were scanned. The mass spectrometry data were collected using the Triple TOF 5600 + Liquid mass spectrometry system (ABSCIEX, Redwood City, CA, USA). The peptide samples were dissolved in 2% acetonitrile/0.1% formic acid and analyzed using a Triple TOF 5600 Plus mass spectrometer coupled with the Eksigent nanoLC System (ABSCIEX, Redwood City, CA, USA). The polypeptide solution was added to a C18 capture column (3 μm, 350 μm × 0.5 mm, AB Sciex, Redwood City, CA, USA) at a time gradient of 60 min. The gradient elution was performed on a C18 column (3 μm, 75 µm × 150 mm, Welch Materials, Inc., Hefei, China) at a flow rate of 300 nL/min. The two mobile phases were buffer A (2% acetonitrile/0.1% formic acid/98% H₂O) and buffer B (98% acetonitrile/0.1% formic acid/2% H₂O). For IDA (information-dependent acquisition), the primary mass spectrometry was performed at 250 ms ion accumulation time, and secondary mass spectrometry of 30 precursor ions was collected at 50 ms ion accumulation time. The MS1 spectra were collected in the range of 350–1200 m/z, and the MS2 spectra were collected in the range of 100–1500 m/z. The dynamic removal time of the precursor ions was set to 15 s.

Peptide samples on the machine. The mass spectrometry data acquisition was performed using a Triple TOF 5600 + liquid mass spectrometry system (AB SCIEX, USA). Peptide samples were dissolved in 2% acetonitrile/0.1% formic acid and analyzed using a Triple TOF 5600 plus mass spectrometer coupled to an Eksigent nanoLC system (AB SCIEX, USA). The peptide solution was added to a C18 capture column (3 µm, 350 µm × 0.5 mm, AB Sciex, USA) and gradient eluted on a C18 analytical column (3 µm, 75 µm × 150 mm, Welch Materials, Inc) with a 90 min time gradient at a flow rate of 300 nL/min. For IDA (information-dependent acquisition), primary mass spectra were scanned with an ion accumulation time of 250 ms, and secondary mass spectra of 30 precursor ions were acquired with an ion accumulation time of 50 ms. MS1 spectra were acquired in the range of 350–1200 m/z, and MS2 spectra were acquired in the range of 100–1500 m/z. The dynamic exclusion time of precursor ions was set to 15 s.

For LC-MS/MS analyses, the fractionated peptides were dissolved in solvent A (0.1% formic acid in 2% acetonitrile, 98% H₂O), loaded onto a reversed-phase precolumn (20 mm × 100 μm, 5 μm) and separated using a reversed-phase analytical column (150 mm × 75 μm, 3 μm) in an Eksigent nanoLC system (Eksigent, Livermore, CA, USA). The gradient for MS analysis was set as follows: starting at 7 to 20% solvent B (0.1% formic acid in 98% acetonitrile, 2% H₂O) over 24 min, then 20–35% solvent B over 8 min, thereafter increasing to 80% solvent B over 55 min and holding at 80% solvent B for the last 3 min, with a flow rate of 300 nL/min. MS data were collected using information-dependent acquisition mode in a high speed TripleTOF™ 5600plus mass spectrometer (ABSciex, Concord, Canada) coupled with the Eksigent nanoLC system. The mass spectrometer was operated in a manner in which a 0.25 s survey scan (MS) in the mass range of 350–1500 m/z was collected, from which the top 30 ions were selected for automated MS/MS in the mass range of 100–1500 m/z and each MS/MS event consisted of a 0.05 s scan. Once a target ion had been fragmented by MS/MS, its mass and isotopes were excluded for a period of 15 s.

The lipidome was analyzed using Waters ACQUITY UPLC (Waters, Milford, USA) coupled with a Triple TOF 5600 Plus mass spectrometer (AB SCIEX, USA) system. Before LC–MS analysis, the lyophilized samples were reconstituted in the mixed solution including CH₂Cl₂ and MeOH (2:1 v/v) and then diluted in the ACN-MeOH-H₂O solution (65:30:5 v/v/v). Next, 5 µl of the diluted sample was separated on the C8 ACQUITYTM column (100 × 2.1 mm, 1.7 µm), (Waters, Milford, MA, USA). The column temperature was kept 60°C. The elution rate was set 0.3 ml/min. The mobile phase A was ACN:H2O (6:4 v/v) and the mobile phase B, was IPA: ACN (9:1 v/v), both containing 10 mM ammonium acetate. The initial elution gradient began with 50% B, kept for 1.5 min, followed by a linear increase to 85% B at 9.0 min, then reached at 100% B within 0.1 min, and maintained for 1.9 min. Lastly, it returned to 50% B within 0.1 min and held for 1.9 min to equilibrate column. The scanned m/z range of MS signal was 200–1,250 Dalton in both positive and negative ion modes. The capillary voltages of the positive and negative ion modes were set at 5.5 and −4.5 kV respectively. The interface heater temperature was set at 500 and 550°C for positive and negative ion modes, respectively.