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.