title: Analysis of acidic peptides with a matrix-assisted laser desorption/ionization mass spectrometry using positive and negative ion modes with additive monoammonium phosphate.
abstract: Acidic PTMs such as phosphorylation and sulfonation of proteins are known to play important roles in many cellular processes including signal transductions and protein-protein interactions. In MS, the acidic modified peptides, that have negative charge, are observable in negative ion mode rather than in positive ion mode. Moreover, addition of ammonium salt into MALDI matrix solution improves the relative intensity of ionization of the phosphorylated peptide to unmodified one. We demonstrate that a combination of the negative ion mode and addition of ammonium salt is more effective in the ionization of the acidic modified peptides. We applied this method to 2-DE separated proteins of Caenorhabditis elegans. As a result, 42 spots were identified as modified proteins, of which 34 proteins were nonoverlapping unique proteins. Furthermore, our study revealed that pI shifts of the DIM-1 and MLC-1 proteins in the 2-DE gel were attributed to the presence of the acidic modifications. The negative ion mode together with the addition of ammonium salt provides us a useful method to detect the phosphorylation and/or sulfonation of protein in a simple manner.
authors.affiliation: Proteomics Research Laboratory, Tokyo Rikakikai Co., Ltd., Tsukuba, Ibaraki, Japan.
authors.forename: Takuji
authors.initials: T
authors.lastname: Nabetani
authors.forename: Kenji
authors.initials: K
authors.lastname: Miyazaki
authors.forename: Yo
authors.initials: Y
authors.lastname: Tabuse
authors.forename: Akira
authors.initials: A
authors.lastname: Tsugita
mesh_terms: D029742:Caenorhabditis elegans Proteins / Q000032:analysis* / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D007477:Ions / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D029742:Caenorhabditis elegans Proteins; D007477:Ions; D010710:Phosphates; D010748:Phosphopeptides; C024788:ammonium phosphate