Microproteins, encoded by small open reading frames (sORFs) have been historically overlooked and are rapidly reshaping our understanding of the proteome. However, large-scale, and confident identification of these microproteins has been technically challenging, compounded by the largely unknown functions of these entities. To address this issue, we have customized sample preparation and data analysis workflows and identified functionally important microproteins in cancer.
First, we compared more than ten methods and reported that size-exclusion chromatography (SEC) could simultaneously enrich and fractionate microproteins from complex proteomes, enabling the discovery of more AltProts with higher overall intensities.
Next, after a comprehensive comparison of mass spectrometry methods and software, we designed a Data-Independent Acquisition (DIA) workflow that leverages a fragmentation spectra predictor for the efficient construction of DIA libraries for microproteins. Our method achieved a two-fold increase in the identification of microproteins and a 50% reduction in missing values compared to traditional methods.
Using this optimized approach, we detected and quantified over a thousand microproteins from hepatocellular carcinoma (HCC) cells. Among them, a novel 54-amino acid microprotein named SEP6, encoded by a lncRNA, was significantly down-regulated in drug-resistant HCC. In both in-situ cell assays and in-vivo animal tests, upregulation of SEP6 restored the sensitivity of HCC and lung cancer cells to multiple drug treatments, including Lenvatinib and regorafenib. Conversely, the knockdown of SEP6 induced drug resistance. Mechanistically, we found that SEP6 interacted with a phosphate, increasing the degradation of g-pg and subsequently enhancing the intracellular concentration of drug molecules.
Our work not only provides a robust workflow from sample preparation to mass spectrometry analysis but also highlights the transformative potential of microprotein research. By uncovering the role of SEP6 in drug sensitivity, we demonstrate the critical impact of microproteins on cancer treatment, paving the way for new therapeutic strategies and enhancing our understanding of the proteome.
Reference:
[1] Yang Y, Wang HW, Zhang YL, Chen L, Chen GL, Bao ZS, Yang Y, Xie Z, Zhao Q*. Molecular & Cellular Proteomics, 2022, 22, 100480
[2] Zhang YL, Chen L, Wang HW, Xie Z, Zhao Q*. Unpublished
[3] Chen L, Yang Y, Zhang YL, Wong C, Lee KW, Zhao Q*. Unpublished