Ultimate 3000 nanolc

All LC‐MS/MS analyses were performed on a system consisting of an Ultimate 3000 nanoLC (Thermo Scientific) coupled via a nanospray interface to a Q Exactive Plus mass spectrometer (Thermo Scientific).

Analyses were performed by injecting 2 µg in quadruplicate into the mass spectrometry platform consisting of an Ultimate 3000 nano LC connected to an Orbitrap Fusion Tribrid MS (Thermo Fisher Scientific, Waltham, MA, USA). Peptides were eluted over two hours by mixing buffer A (99.9% water, 0.1% formic acid) with increasing concentrations of buffer B (99.9% acetonitrile, 0.1% formic acid). This universal method was used with the settings described [36 (link)].
A total of 88 raw data files were entered into MaxQuant version 1.6.6.0 for LFQ analysis [37 (link)] and searched against the Uniprot Sus scrofa database and the Homo sapiens database downloaded on 8 November 2020. Generally, the default settings were used in MaxQuant, including a false discovery rate (FDR) of 0.01 for protein identification and peptide spectrum matches. Digestion with Trypsin was used instead of Trypsin/P, an LFQ minimum ratio count was set to 1, and the match between the runs function was used.

Peptide separation and detection were performed using an Ultimate 3000 nano LC (Thermo Fisher Scientific, San Jose, CA, USA) and an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The system was operated in data-dependent acquisition mode with XCalibur software (ver. 2.0.7, Thermo Fisher Scientific, San Jose, CA, USA). The peptides were loaded onto a C18 PepMap trap column (300 µm ID × 5 mm, Dionex, Thermo Fisher Scientific, San Jose, CA, USA). The peptides were eluted with a linear acetonitrile gradient (8–30% over 150 min) in 0.1% formic acid in acetonitrile at a flow rate of 200 nL/min and were loaded and separated on a C18 capillary tip column (75 µm ID × 120 mm, nano LC capillary column, NTTC-360/75-3, Nikkyo Technos, Tokyo, Japan) with a spray voltage of 1.5 kV. Elution was performed with a linear acetonitrile gradient (5-25% in 120 min) in 0.1% formic acid. Full-scan mass spectra were acquired in the Orbitrap over 400-1,500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy [47 (link)]. The top ten most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at a normalized collision energy of 35%. Dynamic exclusion was employed within 90 s to prevent the repetitive selection of the peptides [48 (link)].

Identification and quantification of the peptides generated from the colorectal tissues were implemented under LC MS/MS system on Ultimate 3000 nanoLC coupled with a Q Exactive HF mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA). To generate the spectrum library, the MS/MS signals were acquired in the data-dependent acquisition (DDA) mode at the following parameter settings: full-scan MS spectra (350–1,500 m/z) with a resolution of 120,000, HCD with 28% relative energy, and MS/MS scan at 15,000 resolution. To quantify the peptide quantities, MS/MS signals were obtained in the DIA mode at the following parameter settings: full-scan MS spectra (350–1,500 m/z) with a resolution of 120,000, HCD with 28% relative energy, fragmentation of precursor ions in sequential windows of 25 m/z, and MS/MS scan at 30,000 resolution. For global calibration of the retention time of peptides, iRT peptides (Ki-3002-1, BIOGNOSIS, Switzerland) were spiked into the digestive peptides at a ratio of 1:100. A peptide mixture was loaded onto a self-packed column (30 cm × 150 μm with 1.8 μm C18 resin) and eluted with a 120-min gradient. The eluted peptides were injected into the mass spectrometer through a nanoelectrospray ionization (ESI) interface.

LC–MS/MS analyses were conducted on a Dionex UltiMate3000 nanoLC system coupled with a Thermo Scientific Q-Exactive HFX or a Fusion Lumos mass spectrometer. Before injection, peptide samples were reconstituted in 2% acetonitrile (ACN), 0.1% formic acid (FA) in LC–MS grade water and centrifuged to collect supernatant. Easy-spray PepMap C18 columns (2 µm, 100 Å, 75 µm × 75 cm) were used for peptide separation with a flow rate of 0.2 µL/min and column temperature of 60 °C. The mobile phase buffer A was 0.1% FA in water, and buffer B was 0.1% FA in acetonitrile. A two-hour gradient was used for proximity labeling proteomics, and a three-hour gradient was used for SILAC proteomics. LC–MS/MS analyses were conducted with a top 40 data dependent acquisition with MS range of m/z 400–1500, MS resolution of 120 K, isolation window of m/z 1.4, dynamic exclusion of 22.5 s, and collision energy of 30% for higher-energy collisional dissociation (HCD) fragmentation. Automatic gain control (AGC) targets were 1 × 10⁶ for MS and 2 × 10⁵ for MS/MS. Maximum injection times (maxIT) were 30 ms for MS and 35 ms for MS/MS. Targeted proteomics was conducted in the parallel reaction monitoring (PRM) mode using an inclusion list generated based on untargeted DDA data with an isolation window of m/z 1.4, retention time window of 5 min, and maxIT of 100 ms.

Quantification
and assay development were performed on an Ultimate 3000 nano-LC (Thermo
Fisher Scientific) connected to an EASY-Spray ion source and a TSQ
Altis (Thermo Fisher Scientific) mass spectrometer. Samples were loaded
on an Acclaim PepMap 100 trap column (75 μm × 2 cm, C18,
3 μm, 100 Å, Thermo Scientific) and washed for 0.75 min
at 15 μL/min with 99% solvent A (3% acetonitrile, 0.1% formic
acid (FA), H₂O). The peptides were separated using an analytical
PepMap RSLC C18 column (150 μm × 15 cm, 2 μm, 100
Å, Thermo Fisher Scientific). Peptides were eluted at a linear
gradient of 1–40% solvent B (95% acetonitrile, 0.1% FA) during
assay development and a linear gradient of 1–30% solvent B
during protein quantification. The flow rate was set to 3 μL/min
over 9.25 min during assay development and over 29.25 min during protein
quantification. The columns were washed three times for 30 s with
95% solvent B followed by 1% solvent B. The columns were equilibrated
for 1.4 min with 1% solvent B. The total turnaround time with sample
loading, analysis, and re-equilibration was 15 min for method development
and 35 min for plasma quantification. The column oven temperature
was maintained at 40 °C, the analytical column was maintained
at 60 °C, and the autosampler temperature was maintained at 10
°C.