New Material Successfully Purifies Cytochrome C for Proteomics


By modifying polyniobate on a metal-organic framework, scientists have managed to purify Cytochrome C for proteomic analysis using LC–MS/MS.

Researchers from Shenyang Medical College in Shenyang, PR China have developed a highly efficient and selective pretreatment method for Cytochrome C (Cyt-C) in real samples. The study, published in the Journal of Chromatography A, describes how polyniobate was modified on a metal-organic framework to create an aqueous-stable composite that can successfully purify complex protein samples (1). The resulting material, Nb6O19/MIL-125(Ti), was found to promote the selective adsorption of Cyt-C due to the synergistic effect of electrostatic and hydrogen-bond interactions.

Cytochrome P450 (CYP2D6) liver enzyme bound to quinine | Image Credit: © -

Cytochrome P450 (CYP2D6) liver enzyme bound to quinine | Image Credit: © -

The study showed that at pH = 10.0, the adsorption efficiency of 300 μL of 100 μg·mL−1 Cyt-C onto 1.0 mg Nb6O19/MIL-125(Ti) can reach 99.5%. The adsorption behavior of Cyt-C fits well with the Langmuir adsorption model, corresponding to a maximum theoretical adsorption capacity of 168.35 mg/g. Using 3 mol/L NaCl as the eluent, a high elution efficiency of 92.19% is obtained.

In other words, Nb6O19/MIL-125(Ti) is a composite material that can selectively adsorb cytochrome C (Cyt-C) due to synergistic electrostatic and hydrogen-bond interactions. This composite material is prepared by modifying polyniobate (K7H[Nb6O19]·13H2O, Nb6O19) on a metal-organic framework MIL-125(Ti). At pH 10.0, Nb6O19/MIL-125(Ti) can efficiently adsorb Cyt-C, with an adsorption efficiency of 99.5% achieved using 1.0 mg of Nb6O19/MIL-125(Ti) for 300 μL of 100 μg/mL Cyt-C. Using 3 mol/L NaCl as the eluent, a high elution efficiency of 92.19% is obtained, making it suitable for the analysis of Cyt-C using LC–MS/MS.

The researchers demonstrated the practical application of Nb6O19/MIL-125(Ti) in scrofa heart proteomics. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed that Nb6O19/MIL-125(Ti) efficiently adsorbed Cyt-C from the scrofa heart extraction. LC–MS/MS spectrometry results show that the purification of Cyt-C reduces the abundance from the 12th to the 154th place after Nb6O19/MIL-125(Ti) treatment. Moreover, low abundant proteins, such as Superoxide dismutase 1, IF rod domain-containing protein, and Ubiquitin-60S ribosomal protein L40, were considerably enriched.

The IF rod domain-containing protein is a type of intermediate filament protein that is involved in maintaining the structural integrity of cells. It is widely distributed in many tissues and is particularly abundant in muscle cells. Ubiquitin-60S ribosomal protein L40 is a ribosomal protein that is involved in the regulation of protein synthesis. It has been shown to play a role in the degradation of misfolded or damaged proteins in cells.

Cyt-C is essential for electron transfer in cellular respiration and its abnormal release from mitochondria to the cytoplasm can cause various diseases. The content of Cyt-C in bio-samples is typically lower than the detection limit of commonly used methods, and complex matrix interference further complicates its analysis. Therefore, the study's success in exploring a new method and material for Cyt-C isolation is of great importance for proteomics. The researchers note that the material could have potential applications beyond scrofa heart proteomics, making it a promising development in the field.


(1) Wu, X.; Mao, Q; Hao, Y; Yang, J.; Zhang, X.; Chi, Z.; Liu, G.; Wang, M; Chen, Q.; Chen, X. Isolation of Cytochrome C for Proteomics with Lindqvist-type Polyiodate Modified Metal Organic Framework. J. Chromatogr. A 2023, 1693, 463869. DOI:

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