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Colorectal Cancer Lines Characterized With Top-Down Proteomics Approaches

NEW YORK – New research has revealed the proteins and protein variants, or proteoforms, found in non-metastatic and metastatic forms of colorectal cancer (CRC) cells, along with proteomic patterns that distinguished them.

"Our study represents a pivotal advance in [top-down proteomics] toward the characterization of [the] human proteome in a proteoform-specific manner, which will transform basic and translational biomedical research," wrote the study's authors led by Liangliang Sun of Michigan State University, Xiaowen Liu at Tulane University, and Amanda Hummon of Ohio State University.

They suggested that such analyses are "crucial for discovering new protein biomarkers and providing a more accurate understanding of molecular mechanisms of cancer metastasis."

As they reported in Science Advances on Wednesday, the researchers used TopPIC analytical software and four distinct capillary zone electrophoresis-tandem mass spectrometry methods with or without size exclusion or liquid chromatography steps for a "top-down proteomics" analysis of non-metastatic and metastatic CRC cell lines generated from the same individual.

While quantitative bottom-up proteomics "usually provides limited information on the proteoforms, which represent all possible protein molecules derived from the same gene resulting from genetic variations, RNA alternative splicing, and protein post-translational modifications," the authors explained, the top-down strategy used for the study "directly measures intact proteoforms and provides opportunities to study functions of specific proteoforms."

From the 2,332 proteins and 23,622 proteoforms found in the cell lines using a false-discovery rate of 1 percent, the team was able to pinpoint proteoform and amino acid variants that distinguished the metastatic form of the disease.

"Quantitative [top-down proteomics] unveiled differentially expressed proteoforms between the two cell lines," the researchers reported, "and the corresponding genes had diversified functions and were closely related to cancer."

By combining data from the mass spec strategies used, the researchers found that the non-metastatic CRC cell line SW480 contained more than 17,300 detectable proteoforms compared to the 14,504 proteoforms identified in the metastatic CRC cell line SW620.

The team took a closer look at the pathways behind the distinct proteoforms found in the mass spec datasets with the help of Qiagen Ingenuity Pathway Analysis.

Both cell lines harbored proteoforms corresponding to WNT/beta-catenin signaling, PI3 kinase, ERK/MAP kinase, and other pathways previously implicated in CRC, the researchers reported, though the number and type of proteoforms from CRC-related pathways and genes varied in the non-metastatic and metastatic cell lines, as did the phosphorylated proteoform and other post-translational modification (PTM) patterns and amino acid variants.

"The data clearly demonstrate that the pair of isogenic human non-metastatic (SW480) and metastatic (SW620) CRC cell lines have substantially different proteoform profiles," the authors wrote.

They noted that "differentially expressed proteoforms, especially those with PTMs, of important cancer-related genes could be novel proteoform biomarkers of CRC metastasis," whereas "proteoforms of genes in well-known CRC-related pathways … are different between SW480 and SW620 cells, and those proteoforms could play vital roles in modulating CRC metastasis."