ALIX protein analysis: storage temperature may impair results

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.