Ptmscan acetyl lysine motif ac k kit

Quantitative proteomics profiling of lysine acetylation was performed by Shanghai Applied protein technology Co., Ltd. Briefly, the sample was dissolved in lysis buffer. The supernatant was collected after centrifugated at 15,000 g for 30 min at 4°C, and the protein concentrations were quantified by Bradfford method. Then, the sample was digested in trypsin, lyophilized, and re-dissolved with 100 mM dissolution buffer for labeling. The TMT reagent was added to digested sample. Subsequently, the labeled samples were incubated with Anti-Ac-K antibody beads [PTMScan Acetyl-Lysine Motif (Ac-K) kit, Cell Signal Technology] for enrichment of acetylated peptides and then analyzed by Easy nLC system coupled with Q-Exactive mass spectrometer. Raw LC–MS/MS data were processed by MaxQuant software (v1.5.3.17). Data were searched against the UniProt Rat complete proteome database of reviewed (Swiss-Prot) and unreviewed (TrEMBL) proteins. From the 2480 AcK-peptides identified, peptides passed the threshold for an adjusted P value ≤ 0.05. Functional enrichment analysis was performed by using KAAS software tool (KEGG Automatic Annotation Server).

A volume (1.4 mL) of pre-cooled immunoaffinity purification (IAP) buffer was used to resuspend each lyophilized peptide sample (3x). The pre-processed anti-Ac-K antibody beads [PTMScan Acetyl-Lysine Motif (Ac-K) Kit, Cell Signal Technology] were added in each tryptic peptide sample, and incubated (1.5h, 4°C). Afterwards, anti-Ac-K antibody beads with acetylated peptides were washed with 1 mL pre-cooled IAP (3x), and with 1 mL pre-cooled water (3x). A volume (40 μl) of 0.15% trifluoroacetic acid (TFA; 302031, Merck) was added to the washed anti-Ac-K antibody beads, and incubated (room temperature, 10 min), and then the same volume of TFA was added once again. The mixture was centrifuged (2000×g, 30s). The supernatant was desalted with C18 STAGE Tips (27 (link)). The desalted supernatant was the enriched acetylated peptide sample.

Freeze-dried peptide samples were redissolved by 1.4 mL of precooled IAP buffer, and then were added pretreated Anti-Ac-K antibody beans (PTMScan Acetyl-Lysine Motif (Ac-K) Kit, Cell Signal Technology). Later incubated at 4 ℃ for 1.5 h, centrifuged at 3000 rpm for 30 s, and the supernatant were discarded. Anti-Ac-K antibody beans were cleaned 3 times with 1 mL precooled IAP buffer and then were cleaned 3 times with 1 mL precooled water again. Added 40 μL 0.15% TFA twice to cleaned anti-Ac-K antibody beans at room temperature for 10 min. And then centrifuged at 3000 rpm for 30 s, and the supernatant was desalinated with C18 STAGE Tips.

Protein (20 mg) was reduced with 10 mM DTT (dithiothreitol) at 37°C for 2 h. After cooling, 50 mM IAA (iodoacetamide) was added for alkylation at room temperature in the dark for 30 min. Add five times the volume of ddH₂O, dilute the urea concentration to 1.5 M, add Trypsin Gold (Promega) at a ratio of 20:1, and digest at 37°C for 16–18 h. After digestion, SPE C18 column (Waters Inc., Milford, MA, United States) was used to peptide desalted and then lyophilized with centrivap vacuum concentrator (Labconco Inc., Kansas City, MO, United States). The lysine-acetylated peptides were enriched by immunoaffinity using anti-Ac-K antibody beads [PTMScan Acetyl-Lysine Motif (Ac-K) Kit, Cell Signal Technology], as previously described (Bouchut et al., 2015 (link)). Briefly, the peptides were mixed with anti-acetyl-lysine beads (Cell Signaling Technology) for 2.5 h at 4°C in MOPS IAP buffer (50 mM MOPS, 10 mM KH₂PO₄, and 50 mM NaCl in 1MTris, pH = 7.0) and then centrifuged for 30 s at 3,000 g at 4°C. Finally, the peptides were eluted with 0.15% TFA. The peptides were desalted using a peptide desalting rotating column (Thermo Fisher Scientific, Germany) before MS identification.

The samples were reconstituted in 1.4 mL of precooled immunoaffinity purification (IAP) buffer (PTMScan IAP buffer; Cell Signaling Technology), pretreated anti-acetylated lysine (Ac-K) antibody beads (PTMScan acetyl-lysine motif [Ac-K] kit; Cell Signaling Technology) were added, the mixture was incubated at 4°C for 1.5 h and centrifuged at 2,000 × g for 30 s, and the supernatant discarded. Anti-Ac-K antibody beads were washed with 1 mL precooled IAP buffer 3 times and then washed with precooled water 3 times. An amount of 40 μL 0.15% TFA was added to the washed beads and incubated for 10 min at room temperature, and then 0.15% TFA was added again, the mixture centrifuged at 2,000 × g for 30 s, and the supernatant desalted by C18 STAGE (stop and go extraction) tips.

After lyophilization, 1.4 ml of precooled immunoaffinity purification buffer (50 mM (3-(N-morpholino) propane sulfonic acid), pH 7.2, 10 mM sodium phosphate, and 50 mM NaCl) was added to the peptide samples for redissolution. Pretreated anti-Kac-antibody beads (PTMScan Acetyl-Lysine Motif (Ac-K) Kit, Cell Signal Technology) were added to the samples. The sample was incubated at 4 °C for 1.5 h and then centrifuged at 2000g for 30 s. The supernatant was discarded. Anti-Kac antibody beads were cleaned three times with 1 ml precooled immunoaffinity purification buffer and then cleaned three times with 1 ml precooled water. After cleaning, the anti-Kac antibody beads were added to 40 μl 0.15% TFA, kept at room temperature for 10 min, and the procedure was repeated. After centrifugation at 2000g for 30 s, the supernatant of the samples was taken and desalted by C18 STAGE Tips (17 (link)).

Phosphopeptides were enriched using High-Select™ TiO₂ Phosphopeptide Enrichment Kit (Thermo, #A32993). Briefly, lyophilized peptide samples were resuspended in Binding/Equilibration buffer and applied to the pre-equilibrated TiO₂ Spin Tip. After washing, enriched phosphopeptides were eluted using the Phosphopeptide Elution Buffer. Then, the eluates were dried immediately in a speed vacuum concentrator to remove elution buffer. For acetylated peptide enrichment, the peptide samples were first dissolved in IPA buffer and incubated with prewashed anti-acetylation antibody beads (PTMScan® Acetyl-Lysine Motif [Ac-K] Kit, Cell Signaling Technology, #13416) at 4°C for 1.5 h incubation. Then the beads were washed three times with IPA buffer and three times with ddH₂O. The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid (TFA), and then, dried by vacuum concentrator. Finally, enriched acetylated peptides were desalted with a Sep-Pak C 18 cartridge and vacuum-dried again.