HCP profile analysis using mass spectrometry
Protein (1.5 mg) from HCCF and ProA pool was diluted in 50 mM ammonium
bicarbonate (pH 8.0) to 1 mg/ml final concentration. Trypsin was added
into the solution at the ratio by weight of 50:1 (substrate to trypsin),
and incubated overnight at 37oC. After adding 7.5 mM
dithiothreitol, the digested samples were boiled at
90oC for 10 min. They were then cooled down rapidly
and precipitates were removed by centrifugation at 16000 x g for 5 min.
The remaining supernatants were applied to Pierce™ Peptide desalting
spin columns (Thermo Scientific, Rockford, USA).
Digested peptides were separated using a ACQUITY UPLC CSH C18 column
(130 Å 1.7 μm, 2.1 x 150 mm) on an ACQUITY Arc high-pressure liquid
chromatography (HPLC) (Waters, Milford, USA). The column was held at
60°C. A 200-min gradient from 98/2 to 55/45 water/acetonitrile (0.1%
formic acid added) was used with a flow rate of 0.3 mL/min. The column
eluate was analyzed on a Thermo Q Exactive Plus mass spectrometer
(Thermo Scientific, Sunnyvale, CA) using DDA mode where the top 20 most
abundant peptide ions were subjected to MS/MS analysis (dynamic
exclusion 60 second, resolution 17500) after a survey scan (m/z range:
300-2000, resolution: 70000). Data analysis was performed by Proteome
Discoverer 2.2 (Thermo Scientific, Sunnyvale, CA) using SEQUEST
algorithm against CHO K1 proteome database (GCF_000223135.1 CriGri_1.0
from www.ncbi.nlm.nih.gov) by adding
decoys and common contaminants. The XCorr confidence thresholds were set
as 1.2, 1.9, 2.3, and 2.6 for charge state z = 1, 2, 3 and z ≥ 4
individually. The delta Cn was set as 0.05, and the
target false discovery rate (FDR) was set as 0.01. The searched results
were further filtered by criteria such as unique pep # ≥ 2 and FDR
Confidence=High. The semi quantitative label-free quantification was
performed using MS peptide signals where the Minora algorithm performed
untargeted feature detection.