10th Annual Congress of the European Proteomics Association, İstanbul, Turkey, 21 - 25 June 2016, pp.1
Differentiation of protein species of alpha-2u-globulin according to database entries
Sevil AKSU, Fatmagül TANRIKULU
Looking into the NCBI protein database there are 35 entries for the protein alpha-2u-globulin of Rattus norvegicus, without entries of fragments. These 35 entries include only thirteen different sequences. Among these entries there are up to 45 varying amino acids in one sequence.
In this approach we digested these 13 sequences theoretically by trypsin and received a list of 51 peptide sequences and masses without missed cleavages. Without the signal sequence but with the allowed modifications of oxidation of methionines and carbamidomethyl-modifications of cysteines the list arose to 72 peptide masses between 700-3000 Daltons [1].
In this investigation we separated the urine proteins of Wistar type adult male rats by 2-DE and identified 19 spots as alpha-2u-globulin by MALDI-TOF/TOF-MS. By comparing the PMF spectra of these spots identified as alpha-2u-globulin we wanted to find out which sequence would be found in which spot. Some masses were found in all of the PMF spectra. There are a few discriminating peptides which can help to distinguish between different sequences or different entries.
The different sequences and thus the different entries for the same protein can be seen as different protein species of one protein. Protein species are defined as “the smallest unit of the proteome” in 1996 and as “chemically the smallest unit, which can be correlated to a function” in 2008 by Junbglut et al. [2, 3].
It could be shown that mass peaks in the PMF spectra of differently positioned spots could be assigned to different sequences/different entries of the same protein, whereas the database searches lead us to one or only a few entries. Since not all existing peptides were sequenced by MS/MS analysis, it was impossible to limit the selection of protein species for each spot. It could be shown that groups of spots contain different peptide sequences.
Keywords: Alpha-2u-globulin, MALDI-MS/MS, NCBI database entries, Protein species, Proteomics
[1] S. Aksu, F. Tanrikulu, Journal of Proteomics 134 (2016) 186–192.
[2] P. Jungblut, B. Thiede, U. Zimny-Arndt, E.C. Muller, C. Scheler, B. Wittmann-Liebold,
A. Otto, Electrophoresis 17 (1996) 839–847.
[3] P. Jungblut, H.G. Holzhütter, R. Apweiler, H. Schlüter, Chem. Cent. J. 2 (2008) 16.