Q exactivetm plus

Control or PPARδ-knockdown HCT116 cells were collected and sonicated on ice in lysis buffer (8 M urea, 1% protease inhibitor cocktail) with a high-intensity ultrasonic processor (Scientz). The supernatant was then harvested, and the concentration of protein was evaluated by a BCA kit following the instructions of manufacturer. After trypsin digestion and labelled by tandem mass tag/isobaric tag for relative and absolute quantitation (TMT/iTRAQ), the tryptic peptides were isolated with an EASY-nLC 1000 UPLC system. Then the peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo Fisher Scientific, USA) coupled online to the UPLC. Next, the acquired MS/MS data were analyzed with the MaxQuant search engine (v.1.5.2.8). Finally, Tandem mass spectra were then searched against the human UniProt database which was concatenated with a reverse decoy database. The minimum score for modified peptides was set at > 40, and FDR was adjusted to < 1%.

To identify the binding proteins of PD-L1, ASPC1 cells were transfected with pcDNA3-Flag vector, Flag-tagged PD-L1. Lysates were immunoprecipitated with Flag-agarose, and then binding proteins were eluted with 2% SDS. Immunoprecipitates were digested with trypsin at 37 °C overnight. Peptides were extracted with 50% acetonitrile/5% formic acid, followed by 100% acetonitrile. Peptides were dried to completion and resuspended in 2% acetonitrile/0.1% formic acid. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive^TM Plus (Thermo) coupled online to the UPLC. GO enrichment analysis was performed using KOBAS.

Trypsin peptides are dissolved in 0.1% formic acid and directly loaded on a self-made reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient of solvent B (0.1% formic acid in 98% acetonitrile) was gradually increased from 6% to 23% through 26 min, increased from 23% to 35% over 8 min, climbing to 80% in 3 min, and then maintained at 80% for 3 min, all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system. The separated peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive^TM Plus (Thermo) coupled online to the UPLC. A 2.0 kV electrospray voltage was applied. At 70,000 resolutions, the intact peptides were detected in the Orbitrap, with 350–1800 m/z full scan range. Up to 20 most abundant precursors were then selected for further MS/MS analyses with 30-s dynamic exclusion. The HCD fragmentation was performed at a normalized collision energy of 28%. The fragments were detected in the Orbitrap at a resolution of 17,500. The fixed first mass was set as 100 m/z. The automatic gain control target was set at 5E4, with an intensity threshold of 1E4 and a maximum injection time of 200 ms.

The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15-cm length, 75 μm i.d.). The gradient was comprised of an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system.
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z^{19 (link),20 (link)}.

The trypsin peptides were resolved in 0.1% formic acid (solvent A) and directly loaded onto a self-made reversed-phase analytical column. The gradient increased from 6% to 23% with 0.1% formic acid in 98% acetonitrile (solvent B) over 26 min. The sample peptides were treated with an NSI source, followed by tandem mass spectrometry (MS/MS) in Q Exactive TM Plus (Thermo) coupled online to the UPLC.

Human vitreous humor pretreatment and TMT analysis were performed as previously described [45 (link), 46 (link)]. Peptide was reconstituted in 0.5 M TEAB, processed according to the manufacturer’s protocol for the TMT kit/iTRAQ kit, and fractionated by high pH reverse-phase HPLC using a Thermo Betasil C18 column (5-μm particles, 10 mm ID, 250 mm length). To enrich succinylation-modified peptides, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) were incubated with prewashed anti-pan-succinylation beads (PTM Bio, PTM-402) at 4 °C overnight with gentle shaking. For LC–MS/MS analysis, the resulting peptides were desalted with C18 ZipTips (Millipore, USA) according to the manufacturer’s instructions. The peptides were subjected to NSI source, followed by tandem mass spectrometry (MS/MS) on a Q ExactiveTM Plus (Thermo Fisher, USA) coupled online to UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350–1800 for the full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. The resulting MS/MS data were processed using the MaxQuant search engine (v.1.5.2.8). Gene Ontology (GO) analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) against the background of Homo sapiens.

The liquid phase A (0.1% formic acid (Fluka)) was used to dissolve the enriched phosphopeptides and load onto a home-made reversed-phase analytical column (length: 15 cm, i.d.: 75 μm), and separated by EASY-nLC 1000 ultra-performance liquid chromatography (UPLC) system. Liquid phase B contains 0.1% formic acid in 90% acetonitrile (Fisher Chemical). The flow rate was maintained at 300 nL/min and the liquid phase gradient setting was as follows: 0–35 min, 4–16% B; 35–65 min, 16–24% B; 65–80 min, 24–40% B; 80–82 min, 40–80% B; 82–90 min, 80% B.
Peptides were subjected to NSI ion source (electrospray voltage: 2.0 kV) to ionize followed by MS/MS in Q Exactive^TM Plus (Thermo), and Orbitrap was used for detection and analysis. The scan range of primary MS was 350 to 1800 m/z at a resolution of 70,000. Then, peptides were selected for MS/MS using NCE setting as 30 and detected with scan range starting at 100 m/z at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied with 15.0 s dynamic exclusion. Automatic gain control (AGC) was set at 1E5.