Xbridge beh130 column

Destained gel was cutted into 10 pieces and digested overnight with 5 μL trypsin (10 ng/μL) at 37°C. For peptide extraction, the trypsin digestions were extracted using filter aided sample preparation (FASP) per the manufacturer's protocol [40 (link)]. The extracted peptide samples were reconstituted with 600 μL of 20 mM ammonium formate at pH10.0, and directly injected into a C₁₈ Waters XBridge BEH130 column (2.1 × 150 mm, Waters, Milford, MA) containing 3.5 μm particles. Peptides separation was performed in a Liquid Chromatography for gradient elution using acetonitrile plus 20 mM ammonium formate at a flow rate of 230 μL/min. As to the MS analysis, peptides were analyzed by a LTQ-OrbitrapVelos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipping with a Nanospray Flex Ion Source (Thermo Fisher Scientific, USA). Full scan MS spectra (m/z 350–2000) was monitored with a resolution of 60,000 at m/z 400. Finally, sequence analysis was carried on the Uniprot human protein database (http://www.uniprot.org/, release 3.43, 72,340 sequences) and database retrieval was performed on the Andromeda search engine based on probabilistic scoring [41 (link)].

An equal amount of peptide was taken separately for each sample, and the peptides were labeled using a TMT10plex labeling kit (Thermo Fisher Scientific, Shanghai, China), and each aliquot (100 μg of peptide equivalent) was reacted separately with a tube of TMT reagent. After dissolving the sample in 100 μL 0.05 M triethyl ammonium bicarbonate (TEAB) solution (pH 8.5), the peptide was labeled using the TMT kit. After labeling, the proteins were fractionated on an Agilent 1290 HPLC using a Waters XBridge BEH130 column (C18, 3.5 μm, 2.1 × 150 mm) at a flow rate of 0.3 mL/min. The pH of two buffers (buffer A: 10 mM ammonium formate, buffer B: 10 mM ammonium formate, and 90% acetonitrile) was adjusted to 10 with ammonium hydroxide. Finally, the samples were collected and combined into 10 components. The peptides of each component were dried and redissolved with 0.1% formic acid (FA) for LC-MS analysis.