TITLE: LC-MS3 Proteomics Data Acquisition
AUTHORS: Ruiqi Jian, Joanne Chan, lihua jiang

[DESCRIPTION]
This protocol described the LC, Mass spectrometry and database search methods used to acquire quantitative proteomic data. For peptides separation, we used a high-low pH 2D LC methods set up for 12 fractions. A MS3 mass spectrometry method was used for TMT10plex  data acquision. Detailed paramters for database search  was described as well.  

[STEPS]
1. Proteomic samples were analyzed on a Thermo Scientific Orbitrap Fusion Lumos  mass spectrometer coupled online with a Waters 2D liquid chromatography (Waters MClass 2DnLC). 
SECTION: Liquid Chromatography Sepration
2. Peptides were separated by reverse phase chromatography at high pH in the first dimension, followed by an orthogonal separation at low pH in the second dimension. 
SECTION: Liquid Chromatography Sepration
3. In the first dimension, the mobile phases were buffer A: 20mM ammonium formate in water at pH10 and buffer B:  100% acetonitrile. Peptides were separated on a Waters Xbridge C18 column (300µm x 5 cm, 5.0µm) using 12 discontinuous step gradient of buffer B: 10.8%, 13.1%, 14.9%, 16.7%, 17.7%, 18.9%, 19.9%, 20.4%, 22.2%, 25.8%, 28.9%, 45% at 2 µl/min flow rate. 
SECTION: Liquid Chromatography Sepration
4. In the second dimension, the mobile phases were buffer A: 0.1% formic acid in water and buffer B: 0.1% formic acid in acetonitrile. An Waters Acquity UPLC M-class Trap 2D Symmetry C18 column (5µm, 180µmx20mm) and an in-house packed analytical column ( 360µm OD/100µm ID/15µm tip ID x 28cm length), packed with Sepex GP-C18 resin ( 1.8um, 120A) were used. Peptides were first trap on trap column , then seperated by analytical column with a gradient from 5% to 40% buffer B at a flow rate of 600 nl/min in 180 minutes. 
SECTION: MS3 Data Accquisition
6. The mass spectrometer was run in a data dependent mode. Full MS scan was acquired in the Orbitrap mass analyzer from 400-1500 m/z with resolution of 120,000.Precursors were isolated with an isolation window of 0.7 m/z and fragmented using CID at 35% energy in ion trap in rapid mode. AGC target was 1e4 and the maximum injection time was 100ms. 
SECTION: MS3 Data Accquisition
5. Analytical column was heated at 45 °C .  Eluted peptides were ionized in positive mode via flex ion source. The source was operated at 1.8-2.2 kV with the ion transfer tube heated at 275 °C. 

SECTION: MS3 Data Accquisition
7. The top 8 fragment ions were selected for MS3 analysis, isolated with an m/z window of 1.6, and fragmented with HCD at 65% energy. Resulting fragments were detected in the Orbitrap at 60,000 resolution, with a maximum injection time of 150ms or until the AGC target value of 1e5 was reached.
SECTION: Data Processing and Analysis
8. The acquired raw data  were processed with the Proteome Discoverer 2.1 (Thermo). 
SECTION: Data Processing and Analysis
9. Mass tolerance of 10ppm was used for precursor ion and 0.6 Dalton for fragment ions for the database search. The search included cysteine carbamidomethylation as a static modification. Acetylation at protein N-terminus, methionine oxidation and TMT at peptide N-terminus and Lysine were used as dynamic modifications. Up to two missed cleavages were allowed for trypsin digestion. Only unique peptides with minimum 6 amino acid length were considered for protein identification. The peptide false discovery rate (FDR) was set as less than 1%. Spectra with more than 50% interference were excluded for quantitative analysis.