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Journal of Molecular and Clinical Medicine   2019, Vol. 2 Issue (2): 29-34    DOI: 10.31083/j.jmcm.2019.02.7161
Original Research Previous articles | Next articles
ALIX protein analysis: storage temperature may impair results
Lopes-Rodrigues Vanessa1, 2, 3, P. R. Xavier Cristina1, 2, Sousa Diana1, 2, 4, Osório Hugo1, 5, 6, G. Assaraf Yehuda7, T. Lima Raquel1, 2, 5, Helena Vasconcelos M.1, 2, 4, *()
1 i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
2 Cancer Drug Resistance Group, IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, 4200-465 Porto, Portugal
3 Institute of Biomedical Sciences Abel Salazar, ICBAS-UP–Institute of Biomedical Sciences Abel Salazar of the University of Porto, 4099-003 Porto, Portugal
4 Department of Biological Sciences, FFUP-Faculty of Pharmacy of the University of Porto, 4050-313 Porto, Portugal
5 Department of Oncology, FMUP–Faculty of Medicine of the University of Porto, 4200-319 Porto, Portugal
6 IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, 4200-465 Porto, Portugal
7 The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, 3200000 Haifa, Israel
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Abstract  

ALIX [ALG-2 (apoptosis-linked gene 2)-interacting protein X] is one of the most well-known molecular biomarkers of extracellular vesicles. Extracellular vesicles are very small vesicles released by most cells and carry in their cargo components from the donor cells, thus being potent vehicles of intercellular (horizontal) communication, influencing various physiological and pathological functions of both recipient and donor cells. The increasing interest in extracellular vesicles highlights the key importance of a reliable analysis of this protein. However, several recent studies in the extracellular vesicles field have shown discrepancies in terms of the expression pattern of apoptosis-linked gene 2-interacting protein X upon Western blot analysis, differing from the theoretical expression pattern of apoptosis-linked gene 2-interacting protein X and its predicted molecular mass. Therefore, to address and clarify this point, we analyzed total protein cell lysates from a chronic myeloid leukemia cell line (K562) for the expression of apoptosis-linked gene 2-interacting protein X by Western blot and mass spectrometry analyses, using protein samples stored at different conditions regarding freezing temperature and storage time. We found that, when stored at -20 oC, a C-terminal specific proteolytic cleavage of apoptosis-linked gene 2-interacting protein X may occur, which depends on the length of storage time. We conclude that analysis of apoptosis-linked gene 2-interacting protein X protein expression should be only carried out when using a wide range of protease inhibitors during isolation of protein cell extract, while preferentially using fresh protein cell extracts or samples that were snap frozen in liquid nitrogen and stored at -80 oC. The current study highlights the importance of proper handling and storage of protein cell lysates for downstream applications in pre-clinical or clinical studies.

Key words:  ALIX      extracellular vesicles      protein degradation      reliable protein identification      storage temperature     
Published:  20 April 2019     
*Corresponding Author(s):  Helena Vasconcelos M.     E-mail:  hvasconcelos@ipatimup.pt

Cite this article: 

Lopes-Rodrigues Vanessa,P. R. Xavier Cristina,Sousa Diana,Osório Hugo,G. Assaraf Yehuda,T. Lima Raquel,Helena Vasconcelos M.. ALIX protein analysis: storage temperature may impair results. Journal of Molecular and Clinical Medicine , 2019, 2(2): 29-34.

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https://jmcm.imrpress.com/EN/10.31083/j.jmcm.2019.02.7161     OR     https://jmcm.imrpress.com/EN/Y2019/V2/I2/29

Figure 1.  Western blot analysis of ALIX protein levels in K562 cells following various storage conditions. Fresh total cell protein lysates were immediately stored at: -20 oC , -20 oC with a phosphatase inhibitor cocktail (+ PI) or at -80 oC . Samples were kept frozen for 1, 2, 3 and 6 weeks. (A) Representative Western blot images; (B) Scanning densitometry analysis of the Western blot ALIX bands, represented as mean ± SEM from three in dependent experiments. β-Actin was used as a loading control. * P ≤ 0.05 when comparing protein levels in samples stored at -20 oC (-PI) vs. other storage conditions (- 80 oC or -20 oC + PI).

Figure 2.  Mass spectrometry analysis of the two ALIX bands. The sequences of both the 93 kDa (A) and 75 kDa (B) bands were analyzed and the peptides related to ALIX were detected. The C-terminal 716-745 peptide, which was present in the sequence of the intact 93 kDa protein (A, marked with a box) was absent from the sequence of the 75 kDa protein (B).

Figure S1.  Mass spectrometry analysis of the 93 kDa ALIX band. The analysis of the sequence of the 93 kDa protein is shown in detail (sequences and peaks of the peptides detected).

Figure S2.  Mass spectrometry analysis of the 75 kDa ALIX band. The analysis of the sequence of 75 kDa protein is shown in detail (sequences and peaks of the peptides detected).

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