Quantification of XRCC and DNA-PK proteins in cancer cell lines and human tumors by LC-MS/MS

Background: The x-ray repair cross-complementing (XRCC) proteins and a catalytic subunit of nuclear DNA-dependent serine/threonine protein kinase (DNA-PK) play important roles in cancer biology. Understanding the protein expression levels allows us to reconstruct in vivo functionality and to qualify protein biomarkers. Methods & results: XRCC and DNA-PK proteins in human cancer cells and tumor tissues have been identified and quantified by selected peptides using NanoLC and high-resolution mass spectrometry. The stable isotope-labeled full-length protein XRCC4 ([¹³C₆, ¹⁵N₄]-arginine and [¹³C₆, ¹⁵N₂]-lysine) uses as the internal standard. Conclusion: The assay range is 0.140–450 fmol (coefficient of variation: 25%) for XRCC4 in bovine serum albumen. The quantitative protein expression levels for XRCC and DNA-PK in HeLa, Ramos and HEK-293 cells and tumor tissues (lung and lymphoma) are reported.

Quantitative proteomic MS analysis of biological samples focuses on identifying the proteins present and establishing the abundance of those proteins in the samples [1–3]. The ability to quantify properly identified proteins in biological samples in a comprehensive fashion engenders an enhanced understanding of cellular behavior during development or in response to disease, and can lead to novel biomarker and target discoveries [4–6]. Developing more accurate and cost-effective methods to quantify target proteins and biomarkers has recently garnered increased effort.

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S-Figure 1. Work Flow of Sample Preparation. Cells or human tissue slices were collected and lysed. Samples were normalized for total protein concentration followed by reduction, alkylation and protein digest with trypsin. LC-MS/MS analysis was performed on an AB Sciex 5600 triple TOF.