Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

A combination approach to explore the missing proteins at low abundance with higher hydrophobicity and lower molecular weight  (#958)

yuanliang zhang 1 , zhilong lin 1 , yan ren 1 , siqi liu 1
  1. BGI, Shenzhen, NOT US OR CANADA, China

In the list of missing proteins (MPs) released in 2019, the proteins with higher hydrophobicity and/or with lower molecular weight occupy at the two top rankings. With MPs being shrunk, a question is naturally raised how to identify a MP class that exhibits low abundance in cells or tissues but possesses high hydrophobicity and/or low molecular weight. A further inquiry is which factor is decisive in technology for discovery of such MPs. Herein, we proposed a combination strategy that was likely as a partial solution for identification of those MPs. The membrane fractions from four cancer cell lines were isolated by ultracentrifuge and the correspondent proteins were equalized in their abundance by ProteoMiner kit. After tryptic digestion of the treated proteins, the resulted peptides were re-extracted by high concentration organic solvent and the extracted peptides were delivered to a mass spectrometer of Orbitrap Fusion™ Lumos™ Tribrid™ for acquiring MS/MS data with high resolution. Based upon the HPP guidelines v 2.10, a total of 16 MPs with 61 unique peptides,in which a MP was defined 2 non-nested unique peptides with ≥9 amino acids at least, were identified in the four cancer cell lines, 2, 8, 2 and 7 MPs in HeLa, HCT116, SNU-1 and HepG2 cells, respectively. The two non-nested unique peptides responding to the individual MPs were further verified through parallel reaction monitoring based upon thechemically synthesized peptides as the standards of retaining time and MS/MS overlay. The combination strategy was thus proven effective in MP discovery.