Zorbax 300sb c18 peptide trap

The two‐dimensional sample was separated using Easy nLC liquid‐phase system. The sample was dissolved with 50 μL solution (0.1% formic acid, 5% acetonitrile) and injected with autosampler into the Zorbax 300SB‐C18 peptide traps (Agilent Technologies, Wilmington, DE, USA). Then, the sample was separated using chromatographic column; the column flow rate is set to 200 nL·min⁻¹. Solution A (0.1% formic acid aqueous solution) and solution B (0.1% formic acid acetonitrile water solution, in which acetonitrile was 100%) were used as the mobile phases with the gradient elution procedures. The liquid‐phase gradient was as follows: 0–100 min, the range of B linear gradient was 5%–30%; 100–130 min, the range of B linear gradient was 30%–40%; 130–135 min, the range of B linear gradient was 40%–90%; and 135–140 min, the range of B was maintained at 90%. Chromatographic column was 75 μm × 150 mm (RP‐C¹⁸), which was equilibrated with 95% solution A.

All of the mass analyses were performed using a nano-LC-MS/MS system, which consisted of a nano-HPLC system (the Ettan MDLC system; GE Healthcare, Piscataway, NJ) and a linear trap quadruple (LTQ) mass spectrometer (LTQ VELOS; Thermo Finnigan, San Jose, CA) equipped with a nano-ESI source. A RP trap column (Zorbax 300SB-C18 peptide traps, Agilent Technologies, Wilmington, DE) was used for desalting of samples, and a C18 reverse-phase column (150 μm i.d., 150 mm length, Column Technology Inc., Fremont, CA) was used for separation. Mobile phase A consisted of HPLC-grade water containing 0.1% formic acid (FA), and phase B consisted of 84% HPLC-grade acetonitrile (ACN) containing 0.1% FA. The analytical separation was run at a flow rate of 2 μl/min by using a linear gradient of phase B as follows: 4%-50% for 105 min, 50%-100% for 9 min and 100% for 6 min. The eluent was then introduced into the LTQ mass spectrometer with the ESI spray voltage set at 3.2 kV. For MS survey scans, each scan cycle consisted of one full MS scan, and five MS/MS events were analyzed. The LC-MS/MS analyses were repeated three times for each independent biological sample. Then the LC-MS/MS results were pooled for each biological replicates to reduce technical variation.

The AA sequences were analyzed by LC-MS/MS. Solution A in the liquid phase was an aqueous solution of formic acid (0.1%), and solution B was an aqueous solution of formic acid–acetonitrile (0.1%, containing 84% acetonitrile). The RP-C18 liquid chromatographic column (0.15 mm × 150 mm, Column Technology Inc., Fremont, CA, USA) was equilibrated with 95% A solution. Additionally, the sample was injected via an autosampler into Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE, USA) for separation on a liquid chromatography column. The liquid phase gradient was set as follows. From 0 min to 50 min, the linear gradient of solution B increased from 4% to 50%. From 50 min to 54 min, the linear gradient of solution B increased from 50% to 100%. From 54 min to 60 min, solution B was maintained at 100%. Mass spectrometry was analyzed using a Q-Exactive mass spectrometer (Thermo Fisher, Boston, MA, USA) in positive ion mode for 60 min. The mass spectrometry data were analyzed by MaxQuant 1.5.5.1, and the UniProt database search was set as follows: MS/MS tolerance was 0.2 Da with up to two missing fragmentations, and the methionine oxidation served as variable modifications. The confidence level for positive protein identification was determined based on the high protein and peptide fractions in the search results.

The tape samples were prepared and lyophilized, and 40 μl of trypsin buffer was added and incubated at 37 °C for 16-18 h. The mobile phase A was 0.1% formic acid aqueous solution and B was 0.1% formic acid acetonitrile aqueous solution. The liquid chromatography column (0.15 mm*150 mm, RP-C18, Column Technology Inc) was equilibrated with 95% A solution, the samples were loaded by an autosampler into Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE), and separated in a liquid chromatography column with a liquid phase gradient: 0–15 min, linear gradient from 4 to 50% for B solution; 15–19 min, linear gradient from 50 to 100% for B solution; 19–20 min, liquid B was maintained at 100%. Subsequent mass spectrometry was performed using a mass spectrometer Q Exactive, analysis time: 20 min, detection: positive ions. The software MaxQuant 1.5.5.1 was used to search the corresponding database for raw files of mass spectrometry assays, and the protein identification and quantitative analysis results were obtained. Based on the identification results, primary structure analysis and secondary and tertiary structure prediction, homology modeling, and crystal structure analysis were performed on the proteins.

The principal components of LAP gel fractions were identified by liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) using a Q Exactive mass spectrometer (Q Exactive, Thermo Fisher, Waltham, MA, USA). The LC-MS/MS method was performed following the methods of Cai et al. [53 (link)] with slight modifications. About 1 mg/mL of LAP principal components was loaded onto an RP-C18 column (Column Technology Inc., 0.15 mm × 150 mm) and balanced with 95% solvent A (0.1% formic acid aqueous solution). The sample solution was then sent to Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, NC, USA) and separated by a liquid chromatography column. The elution program consisted of the solvent B (84% acetonitrile, 0.1% formic acid) concentration increasing from 4% to 50% over a period of 50 min, followed by a concentration from 50% to 100% over a period of 54–60 min. Data analysis was performed using MaxQuant 1.5.5.1 software and the NCBI_Cobitidae database. The screening criteria for reliable identification of peptides were set at p ≤ 0.01.

The liquid A used in the liquid phase was a 0.1% formic acid aqueous solution, and the B liquid was a 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%). A liquid chromatography column (0.15 mm * 150 mm, RP-C18, Column Technology Inc., Fremont, CA, USA) was equilibrated with 95% solution A, and the sample was loaded by an autosampler onto Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE, USA), and then separated by a liquid chromatography column. The relevant liquid phase gradient settings were as follows: 0–50 min, the linear gradient of liquid B was from 4% to 50%; 50–54 min, the linear gradient of liquid B was from 50% to 100%; 54–60 min, solution B was maintained at 100%. The enzymatic hydrolysis products were separated by capillary high performance liquid chromatography and then analyzed by a Q Exactive mass spectrometer (Thermo Fisher). Analysis time: 60 min. The original file of the mass spectrometry test (raw file) was searched with the software MaxQuant 1.5.5.1 to the corresponding database, and finally the peptide chain sequence was obtained.

The peptide sequence of the optimal component for whitening activity was identified using HPLC-MS/MS. An RP-C18 column (0.15 mm × 150 mm, Column Technology Inc., Fremont, CA, USA) was equilibrated with a 95% aqueous solution of 0.1% formic acid. The sample was loaded onto Zorbax 300SB-C18 peptide traps (Agilent Technologies Inc., Wilmington, DE, USA), and then separated using liquid chromatography. The liquid-phase gradient was set as follows: 0–50 min, a linear gradient of 0.1% formic acid in acetonitrile aqueous solution from 4% to 50%; 50–54 min, a linear gradient of 0.1% formic acid in acetonitrile aqueous solution from 50% to 100%; and 54–60 min, 0.1% formic acid in acetonitrile aqueous solution maintained at 100%. A Q Exactive^TM mass spectrometer (Thermo Fisher Scientific Inc., Bremen, Germany) was used for detection in the positive ion mode, and the analysis time was 1 h. The raw data were searched against the corresponding database using the MaxQuant 1.5.5.1 software (Max Planck Institute of Biochemistry., Martins Reid, Germany) and compared with the UniProt database for analysis. Finally, the target peptide sequence and its identification results were obtained.