Comparative proteomic analysis of cysteine oxidation in colorectal cancer patients

Abstract

Oxidative stress promotes damage to cellular proteins, lipids, membranes and DNA, and plays a key role in the development of cancer. Reactive oxygen species disrupt redox homeostasis and promote tumor formation by initiating aberrant activation of signaling pathways that lead to tumorigenesis. We used shotgun proteomics to identify proteins containing oxidation-sensitive cysteines in tissue specimens from colorectal cancer patients. We then compared the patterns of cysteine oxidation in the membrane fractions between the tumor and non-tumor tissues. Using nano-UPLC-MS(E) proteomics, we identified 31 proteins containing 37 oxidation-sensitive cysteines. These proteins were observed with IAM-binding cysteines in non-tumoral region more than tumoral region of CRC patients. Then using the Ingenuity pathway program, we evaluated the cellular canonical networks connecting those proteins. Within the networks, proteins with multiple connections were related with organ morphology, cellular metabolism, and various disorders. We have thus identified networks of proteins whose redox status is altered by oxidative stress, perhaps leading to changes in cellular functionality that promotes tumorigenesis.

Fig. 1.

Detection of proteins containing oxidation-sensitive cysteine residues. (A) Histological staining of tissues from CRC patients. The solid lines, non-tumoral regions; the dotted lines, tumoral regions. (B) Modification of cysteines in the membrane fraction of tumor and non-tumor tissues detected using BIAM-streptavidin immunoblotting. Samples were run in triplicate, and Coomassie-stained proteins served as a loading control. pN, non-tumor tissues of CRC patients; pT, tumor tissues of CRC patients.

Fig. 2.

Detection of proteins containing IAM binding cysteine by immunoprecipitation using BIAM-streptavidin beads. (A) A representative gel image of IAM-binding level of COL6A1, TPSB2 and PDIA3. The cysteine oxidation of COL6A1, TPSB2 and PDIA3 was confirmed in the membrane fraction of normal subject, non-tumoral, and tumoral tissues (see the information in Table 2). Samples were run in triplicate. (B) Histograms of IAM-binidng level of COL6A1, TPSB2, and PDIA3. The graph is shown along with the numeric data obtained by densitometry analysis (n = 5). H, normal tissue of healthy subject; pN, non-tumor tissues of CRC patients; pT, tumor tissues of CRC patients.

Fig. 3.

Reciprocal network annotation among proteins containing oxidation-sensitive cysteines determined using IPA. Candidate proteins containing oxidation-sensitive cysteines (Tables 2 and 3) are shown as gray-filled figures; solid lines, protein-protein interactions; solid arrow lines, expression/membership; dotted arrow lines, expression/activation.