Optimization of Microflow LC Coupled with Scanning SWATH and Its Application in Hepatocellular Carcinoma Tissues

Scanning
SWATH coupled with normal-flow LC has been recently introduced
for high-content, high-throughput proteomics analysis, which requires
a relatively large amount of sample injection. Here we established
the microflow LC coupled with Scanning SWATH for samples with relatively
small quantities. First, we optimized several key parameters of the
LC and MS settings, including C18 particle size for the analytical
column, LC gradient and flow rate, as well as effective ion accumulation
time and isolation window width for MS acquisition. We then compared
the optimized Scanning SWATH method with the conventional variable
window SWATH (referred to as SWATH) method. Results showed that the
total ion chromatogram signals in Scanning SWATH were 10 times higher
than that of SWATH, and Scanning SWATH identified 12.2–22.2%
more peptides than SWATH. Finally, we employed 120 min Scanning SWATH
to acquire the proteomes of 62 formalin-fixed, paraffin-embedded (FFPE)
tissue samples from 31 patients with hepatocellular carcinoma (HCC).
Altogether, 92 334 peptides and 8516 proteins were quantified.
Besides the reported biomarkers, including ANXA2, MCM7, SUOX, and
AKR1B10, we identified new potential HCC biomarkers such as CST5,
TP53, CEBPB, and E2F4. Taken together, we present an optimal workflow
integrating microflow LC and Scanning SWATH that effectively improves
the protein identification and quantitation.