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Titanium(IV) immobilized affinity chromatography facilitated phosphoproteomics analysis of salivary extracellular vesicles for lung cancer

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Abstract

Extracellular vesicles (EVs) play critical roles in intercellular communications, which contain valuable biomarkers for the detection of cancers. Phosphoproteomics analysis of human saliva EVs (sEVs) can help to discover lung cancer–related candidates. Due to the low abundance of phosphoproteins in sEVs, an efficient, reproducible, and cost-effective strategy is required for their enrichment. Here, we compared the latest phosphopeptide techniques, including TiO2, ZrO2, CaTiO3, and Ti4+-IMAC (immobilized metal affinity chromatography) methods, for phosphopeptide isolation. Our data demonstrated that Ti4+-IMAC was the superior one. By using the optimized Ti4+-IMAC approach, we identified more than 500 sEV phosphopeptides. Quantitative proteomics was employed to comprehensively decipher the sEV phosphoproteome of the normal group (n = 6) and lung cancer group (n = 6). Accordingly, 524 and 333 phosphopeptides were enriched, respectively, which corresponded to 439 and 282 phosphoproteins. In total, 857 unique sEV phosphopeptides corresponding to 721 phosphoproteins were revealed. Among 493 identified phosphosites, 37 were upregulated (> 1.5) and 217 were downregulated (< 0.66) in the cancer group. Our data collectively demonstrated that Ti4+-IMAC is an efficient and reproducible technology for comprehensive analysis of sEV phosphoproteome. Differentially expressed sEV phosphoproteins and phosphosites might be used for the detection of lung cancer non-invasively.

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Funding

This work was supported by grants from the Natural Science Foundation of Shanghai (No. 21ZR1433200), the National Key Research and Development Program of China (No. 2017YFC1200204), the National Natural Science Foundation of China (No. 21675110), and the Key Scientific Project of Shanghai Jiao Tong University (No. TMSK-2020–130, No. YG2017MS80).

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Author notes

  1. Amir Wahid and Amir Sohail contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China

    Amir Wahid, Amir Sohail, Huiyu Wang, Miao Guo, Lu Zhang & Hua Xiao

  2. State Key Laboratory of Translational Medicine and Innovative Drug Development, Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing, 210042, China

    Yin Ji & Peng Wang

Authors
  1. Amir Wahid
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  2. Amir Sohail
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  3. Huiyu Wang
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  4. Miao Guo
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  5. Lu Zhang
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  6. Yin Ji
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  7. Peng Wang
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  8. Hua Xiao
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Correspondence to Peng Wang or Hua Xiao.

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In this study, no human patients or animals were involved. Saliva sample were obtained from the Shanghai Jiao Tong University affiliated Chest Hospital according to approved protocols (IRB#M15017) by Institutional Review Board (IRB) of Shanghai Jiao Tong University.

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Wahid, A., Sohail, A., Wang, H. et al. Titanium(IV) immobilized affinity chromatography facilitated phosphoproteomics analysis of salivary extracellular vesicles for lung cancer. Anal Bioanal Chem 414, 3697–3708 (2022). https://doi.org/10.1007/s00216-022-04013-7

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  • DOI: https://doi.org/10.1007/s00216-022-04013-7

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