Session: Session 1

Enhanced proteomics digestion by using hexafluoroisopropanol as an additive

The solubilization of proteins and peptides after digestion is important for proteomic identification. For example, during FASP digestion, sodium deoxycholate (DCA) is used to improve solubilization and identification of peptides and proteins. However, DCA leaves traces in mass spectrometry analysis. The aim is to compare different digestion methods containing solubilizing agents such as DCA, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP).



One microliter of human plasma was digested using either liquid digestion or FASP digestion with tryspin as the digesting enzyme. For digestion methods, several denaturation buffers were tested with TFE, HFIP or DCA. Different enzymatic hydrolysis buffers were tested with or without HFIP or DCA. Peptides were analyzed by nanoESI-LC-MSMS orbitrap Q-Exactive plus using DDA or DIA mode. Proteins were analyzed using Maxquant for DDA mode and DIA-NN for DIA mode. Statistical analysis was performed using Analyst-suites



For liquid digestion, statistical analysis enables us to identify specific proteins when HFIP solubilizing agent was used. The DIA mode providds a greater increase in the number of proteins in human plasma than the DDA mode for both conditions (TFE and HFIP). In DIA we detect 34 significant differentially expressed proteins, 25 of which are overexpressed in HFIP conditions. Furthermore, no contaminant peaks were observed with HFIP in the LC-MSMS analyses. The HFIP method seems to outperform the TFE method. The classical FASP method with DCA outperformed the liquid digestion. However, HFIP did not work on Amicon® filters. To overcome this problem, we are currently testing different filters made from PVDF or PES.



HFIP looks promising for proteomic digestions and LC-MSMS analysis