Three main steps were carried out as follows: protein extraction and peptide digestion, data dependent acquisition (DDA) mass spectrometry assay, DIA mass spectrometry assay and data analysis. The workflow is shown in Figure 1
Plasma pools were separated of most abundant proteins following the manufacturer’s protocol (Agilent Technologies). The high and low abundance proteins were collected, desalted and concentrated respectively. 200 μg proteins was repeat ultrafiltered using UA buffer (8 M urea, 150 mM Tris-HCl pH 8.0). Then 100 μl iodoacetamide (100 mM IAA in UA buffer) was added to block reduced cysteine residues and the samples were incubated for 30 min in darkness. The protein suspensions were digested with 4 μg trypsin in 40 μl 25mM NH4HCO3 buffer overnight at 37°C. Collected peptides were desalted on C18 Cartridges and reconstituted in 40µl of 0.1% formic acid. The iRT-Kits (Biognosys) was added to correct the relative retention time.
All fractions for DDA library generation were analyzed by a Thermo Scientific Q Exactive HF X mass spectrometer connected to an Easy nLC 1200 chromatography system (Thermo Scientific). MS detection method was positive ion, the scan range was 300-1800 m/z, resolution for MS1 scan was 60000 at 200 m/z. Resolution for MS2 scan was 15000. Each DIA cycle contained one full MS-SIM scan, and 30 DIA scans covered a mass range of 350-1800 m/z. QC samples were injected with DIA mode at the beginning of the MS study and after every 6 injections throughout the experiment, which was used to monitor the MS performance.
For DDA library data, the FASTA sequence database was searched with Spectronaut™ software. Spectral library was constructed by importing the original raw files and DDA searching results into Spectronaut Pulsar X™ (Biognosys). DIA data was analyzed with Spectronaut™ 14.4.200727.47784 searching the above constructed spectral library. All results were filtered based on Q value cutoff 0.01 (equivalent to FDR<1%).
Plasma pools were separated of most abundant proteins following the manufacturer’s protocol (Agilent Technologies). The high and low abundance proteins were collected, desalted and concentrated respectively. 200 μg proteins was repeat ultrafiltered using UA buffer (8 M urea, 150 mM Tris-HCl pH 8.0). Then 100 μl iodoacetamide (100 mM IAA in UA buffer) was added to block reduced cysteine residues and the samples were incubated for 30 min in darkness. The protein suspensions were digested with 4 μg trypsin in 40 μl 25mM NH4HCO3 buffer overnight at 37°C. Collected peptides were desalted on C18 Cartridges and reconstituted in 40µl of 0.1% formic acid. The iRT-Kits (Biognosys) was added to correct the relative retention time.
All fractions for DDA library generation were analyzed by a Thermo Scientific Q Exactive HF X mass spectrometer connected to an Easy nLC 1200 chromatography system (Thermo Scientific). MS detection method was positive ion, the scan range was 300-1800 m/z, resolution for MS1 scan was 60000 at 200 m/z. Resolution for MS2 scan was 15000. Each DIA cycle contained one full MS-SIM scan, and 30 DIA scans covered a mass range of 350-1800 m/z. QC samples were injected with DIA mode at the beginning of the MS study and after every 6 injections throughout the experiment, which was used to monitor the MS performance.
For DDA library data, the FASTA sequence database was searched with Spectronaut™ software. Spectral library was constructed by importing the original raw files and DDA searching results into Spectronaut Pulsar X™ (Biognosys). DIA data was analyzed with Spectronaut™ 14.4.200727.47784 searching the above constructed spectral library. All results were filtered based on Q value cutoff 0.01 (equivalent to FDR<1%).
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