Accelerated Protein Biomarker Discovery from FFPE Tissue Samples Using Single-Shot, Short Gradient Microflow SWATH MS

We reported and evaluated a microflow,
single-shot, short gradient
SWATH MS method intended to accelerate the discovery and verification
of protein biomarkers in preclassified clinical specimens. The method
uses a 15 min gradient microflow-LC peptide separation, an optimized
SWATH MS window configuration, and OpenSWATH software for data analysis.
We applied the method to a cohort containing 204 FFPE tissue samples
from 58 prostate cancer patients and 10 benign prostatic hyperplasia
patients. Altogether we identified 27,975 proteotypic peptides and
4037 SwissProt proteins from these 204 samples. Compared to a reference
SWATH method with a 2 h gradient, we found 3800 proteins were quantified
by the two methods on two different instruments with relatively high
consistency (r = 0.77). The accelerated method consumed
only 17% instrument time, while quantifying 80% of proteins compared
to the 2 h gradient SWATH. Although the missing value rate increased
by 20%, batch effects reduced by 21%. 75 deregulated proteins measured
by the accelerated method were selected for further validation. A
shortlist of 134 selected peptide precursors from the 75 proteins
were analyzed using MRM-HR, and the results exhibited high quantitative
consistency with the 15 min SWATH method (r = 0.89)
in the same sample set. We further verified the applicability of these
75 proteins in separating benign and malignant tissues (AUC = 0.99)
in an independent prostate cancer cohort (n = 154).
Altogether, the results showed that the 15 min gradient microflow
SWATH accelerated large-scale data acquisition by 6 times, reduced
batch effect by 21%, introduced 20% more missing values, and exhibited
comparable ability to separate disease groups.