Page 52 - JCBP-2-3

Journal of Clinical and
Basic Psychosomatics Proteomic analysis of mind-body psychotherapy in psoriasis

A B

Figure 1. Proteomics experiment and analysis workflow. (A) Proteomics experiment workflow. (B) Proteomics data analysis workflow
Abbreviations: PPI: Protein and protein interaction; KEGG: Kyoto encylopedia of Genes and Genome.

2.4. Protein digestion mobile phase B rose from 25% to 35%; 105 – 110 min,
mobile phase B rose from 35% to 80%; 110 – 115 min, 80%
The obtained protein-containing solution was incubated
at 37°C for 45 min after added with an appropriate mobile phase B; 115 – 120 min, and 5% mobile phase B.
amount of 10 mM dithiothreitol. Then, it was treated The nanoliter liquid phase separation was directly
with 25 mM iodoacetamide and left in a dark room connected to the mass spectrometer (BGI, Beijing,
30
for 30 min at room temperature. After trypsinization, China). For DDA analysis, LC-separated peptides were
the solution was incubated for 14 – 16 h at 37°C. The ionized by nanoESI. They were injected into a tandem mass
digested peptides for spectral library generation were spectrometer Q-Exactive HF X (Thermo Fisher Scientific,
divided into ten fractions with a C18 StageTip. The San Jose, CA, USA) with DDA detection mode. The main
Shimadzu LC-20AB HPLC system coupled with a settings were as follows: ion source voltage = 1.9 kV; MS scan
Gemini high pH C18 column (5 m, 4.6 × 250 mm) was range = 350–1,500 m/z; MS resolution = 120,000; maximal
used. All peptides were frozen, dried, and desalted before injection time (MIT) = 50 ms; MS/MS collision type = HCD;
liquid chromatography-tandem mass spectrometry collision energy (nominal collison energy) = 28; MS/MS
(LC-MS/MS) analysis (BGI, Shenzhen, China). 30 resolution = 30,000; MIT = 100 ms; and dynamic exclusion
duration = 30 s. The start m/z for MS/MS was fixed at 100.
2.5. Data-dependent acquisition (DDA) and data- A precursor for MS/MS had the following setting: charge
independent acquisition (DIA) analysis by nano- range = 2+ to 6+; top 20 precursors with intensity over 2E4,
LC-MS/MS and automatic gain control (AGC) = MS 3E6, MS/MS 1E5.
The dried peptide samples were reconstituted with mobile For DIA analysis, LC-separated peptides were ionized
phase A (2% acetonitrile [ACN], 0.1% formic acid [FA]), by nanoESI and injected into tandem mass spectrometer
centrifuged at 20,000 ×g for 10 min, and the supernatant Q-Exactive HF X (Thermo Fisher Scientific, San Jose,
was taken for injection. Separation was carried out by a CA, USA) with DIA detection mode. The main settings
Thermo UltiMate 3000 UHPLC liquid chromatography. were as follows: ion source voltage = 1.9 – 2 kV; MS
The sample was enriched in the trap column and desalted scan range = 400 – 1250 m/z; MS resolution = 120,000;
and then entered a tandem self-packed C18 column MIT = 50 ms; and 400 – 1250 m/z was equally divided into
(150 µm internal diameter, 1.8 µm column size, 35 cm a 45 continuous windows of MS/MS scan. MS/MS collision
column length) and was separated at a flow rate of 500 type was collision dissociation (HCD), while MIT was
nL/min by the following effective gradient: 0 – 5 min, 5% on auto mode. Fragment ions were scanned in Orbitrap,
mobile phase B (98% ACN, 0.1% FA); 5 – 90 min, mobile with MS/MS resolution = 30,000, collision energy in the
phase B linearly increased from 5% to 25%; 90 – 105 min, distributed mode (22.5, 25, 27.5), and AGC = 1E6.

Volume 2 Issue 3 (2024) 4 doi: 10.36922/jcbp.2381