Q exactive mass spectrometry

The labeled peptide samples were purified and separated by AKTA purification system. The operation methods and solution preparation were performed essentially as described previously [19 (link)]. The whole elution process was monitored at 214 nm and collected every minute. Thirty distillates were collected and neutralized in 10 pools and desalinated in a C18 cartridge. After each fraction was vacuum centrifuged, the sample was dissolved in 40 μL 0.1% trifluoroacetic acid and kept frozen at − 80 °C for mass spectrometry analysis. Each sample was separated by capillary high-performance liquid chromatography (Thermo scientific EASY column (2 cm, 100 μm 5 μm, C18). The chromatography conditions were as follow: Water with 0.1% formic acid (A) and Acetonitirile with 0.1% formic acid (B) as mobile phase. The flow rate was 300 nL per minute and the mobile phase gradient program was used: 0–33 min, from 0 to 40%(B); 33–34 min, from 40 to 100%(B); 34–35 min maintained 100% and then back to 40%. Then, proteins were analyzed by using a Q-Exactive mass spectrometry (Thermo Finnigan) at positive ion mode (parameters: mass range: 300–1800 m/z; Dynamic exclusion: 40.0 s, MS2 Activation Type: HCD, Normalized collision energy: 30 eV).

Filter-aided sample preparation (FASP) method was used to handle protein samples. Proteins were hydrolyzed to peptides by trypsin digestion, and peptide solution was prepared for further LC–MS/MS analysis. The peptide was separated by a Nano-HPLC (EASY-nLC1200) which has a reversed-phase column (100 μm, ID × 15 cm, Reprosil-Pur 120 C18-AQ, 1.9 um, Dr. Math). Samples were separated at a 300 nl/min flow rate with the following gradient: 0–40 min, 5–30% B; 40–54 min, 30–50% B; 54–55 min, 50–100% B; 55–60 min, 100% B. Mobile phases A and B were H₂O-FA and ACN-H₂O-FA, respectively. Q-Exactive mass spectrometry (ThermoFinnigan) was used for peptide scanning. MaxQuant (Version 1.5.6.0) was used for processing raw MS files. The human protein sequence database (Uniprot_HUMAN_2016_09) was downloaded from UNIPROT.

The hippocampal tissues were homogenized in RIPA buffer containing 2% PMSF and a 1% protease inhibitor cocktail. After quantification with the Pierce™ BCA Protein Assay Kit, proteins were precipitated with acetone. A total of 200 μL of 100 mM ammonium bicarbonate containing 1% SDC was added to re-dissolve the protein pellet. Values of 5 mM tris (2-carboxyethyl) phosphine and 10 mM iodoacetamide were added to alkylate the reduced disulfide bond, and then enzymatically digested with 2 μg trypsin. Trifluoroacetic acid was added to the mixed sample to precipitate SDC. After desalting the peptides, they were separated by the nano-UPLC (EASY-nLC1200, Thermo Fisher Scientific, Waltham, MA, USA) and detected using Q-Exactive mass spectrometry (Thermo Finnigan, Silicon Valley, CA, USA). Raw MS files were processed with MaxQuant (Version 1.5.6.0, Max Planck Institute, Munich, Germany). The quantification type was label-free quantification which was calculated by a label-free, intensity-based absolute quantification approach. Each group of samples contained hippocampal tissues of 6 mice.
When the ratio of protein between the two groups was greater than 1.5 or less than 0.66, they were considered to be proteins with significant differences. The cluster heat map and protein interaction analysis were executed.

Firstly, 1 μg of the peptide was isolated and analyzed by nano UPLC (EASY-nLC1200) and Q Exactive mass spectrometry (Thermo Finnigan). Each component was replicated three times. Separation of peptides was performed by reverse-phase column (100 μm, ID × 15 cm, Reprosil-Pur120 C18-AQ, 1.9 μm) in buffer A (0.1% FA, 2% ACN) and buffer B (0.1% FA, 80% ACN) with a flow rate of 300 nL/min. Linear gradient: 6–28% for 70 min, 28–40% for 12 min, 40–100% for 2 min, 100% for 2 min, 100–2% for 2 min, 2% for 2 min. Next, the mass spectrometry detection was performed under positive ion mode by Q Exactive mass spectrometry (90 min/sample). Data were acquired in DDA mode (20 pieces each time). The standardized collision energy was set to 32%. The isolation window was set to 2 m/z. The dynamic exclusion time was set to 30 s. MS parameters were as follows: (1) Primary mass spectrometry: Scanning range of the parent ions was 350–1600 m/z; Resolution of 70,000 at 200 m/z; The automatic gain control (AGC) target was set to 3E6; Maximum ion injection time (Max IT) was set to 50 ms; (2) Secondary mass spectrometry: Resolution of 17,500 at 200 m/z; AGC target was set to 1E5; Max IT was set to 100 ms.

HASMCs were infected with pAd-GFP, pAd-GFP-KLF4, or pAd-eEF1A2. Then the protein isolation and TMT-based LC-MS/MS analysis were performed by KangChen BioTech (Shanghai). The cells were lysed with RIPA lysis buffer, and the protein was then isolated. The protein content was determined by BCA assay. 100 μg protein for each sample was used for TMT labeling and LC-MS/MS in the following steps: (1) reduction and alkylation; (2) acetone precipitation; (3) re-suspend protein for tryptic digest; (4) TMT labeling; (5) cleaning up of SDC; (6) peptide desalting for LC-MS/MS; and (7) LC-MS/MS: 100 μg peptides were fractionated to 120 fractions with high pH RPRP-HPLC, and then combined to 8 fractions. For each fraction, 2 μg peptides were separated and analyzed with a Nano-HPLC (EASY-nLC1200) coupled to Q-Exactive mass spectrometry (Thermo Finnigan). Proteins were selected based on being significantly differentially expressed (fold changes ≥1.2).

For each sample, ∼2 µg peptides were separated and analyzed with a nano-UPLC (EASY-nLC1200) coupled to Q-Exactive mass spectrometry (Thermo Finnigan, ‎Waltham, MA). Separation was performed using a reversed-phase column (100 µm, ID × 15 cm, Reprosil-Pur 120C 18-AQ, 1.9 µm, Dr. Math). Mobile phases were H₂O with 0.1% FA, 2% ACN (phase A) and 80% ACN, 0.1% FA (phase B). Separation of sample was executed with a 120 min gradient at 300 nL/min flow rate. Gradient B: 8–30% for 92 min, 30–40% for 20 min, 40–100% for 2 min, 100% for 2 min, 100–2% for 2 min and 2% for 2 min.
Data dependent acquisition was performed in profile and positive mode with Orbitrap analyzer at a resolution of 70,000 (@200 m/z) and m/z range of 350–1600 for MS1; for MS2, the resolution was set to 17,500 with a dynamic first mass. The automatic gain control (AGC) target for MS1 was set to 3.0E + 6 with max IT 50 ms, and 5.0E + 4 for MS2 with max IT 100 ms. The top 20 most intense ions were fragmented by HCD.