title: Detection and site localization of phosphorylcholine-modified peptides by NanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring experiments.
abstract: Phosphorylcholine (PC)-modified biomolecules like lipopolysaccharides, glycosphingolipids, and (glyco)proteins are widespread, highly relevant antigens of parasites, since this small hapten shows potent immunomodulatory capacity, which allows the establishment of long-lasting infections of the host. Especially for PC-modified proteins, structural data is rare because of the zwitterionic nature of the PC substituent, resulting in low sensitivities and unusual but characteristic fragmentation patterns. We have developed a targeted mass spectrometric approach using hybrid triple quadrupole/linear ion trap (QTRAP) mass spectrometry coupled to nanoflow chromatography for the sensitive detection of PC-modified peptides from complex proteolytic digests, and the localization of the PC-modification within the peptide backbone. In a first step, proteolytic digests are screened using precursor ion scanning for the marker ions of choline (m/z 104.1) and phosphorylcholine (m/z 184.1) to establish the presence of PC-modified peptides. Potential PC-modified precursors are then subjected to a second analysis using multiple reaction monitoring (MRM)-triggered product ion spectra for the identification and site localization of the modified peptides. The approach was first established using synthetic PC-modified synthetic peptides and PC-modified model digests. Following the optimization of key parameters, we then successfully applied the method to the detection of PC-peptides in the background of a proteolytic digest of a whole proteome. This methodological invention will greatly facilitate the detection of PC-substituted biomolecules and their structural analysis.
authors.affiliation: Protein Analytics, Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University Giessen, 35392, Giessen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Timm
authors.forename: Christof
authors.initials: C
authors.lastname: Lenz
authors.forename: Dietrich
authors.initials: D
authors.lastname: Merkel
authors.forename: Christian
authors.initials: C
authors.lastname: Sadiffo
authors.forename: Julia
authors.initials: J
authors.lastname: Grabitzki
authors.forename: Jochen
authors.initials: J
authors.lastname: Klein
authors.forename: Guenter
authors.initials: G
authors.lastname: Lochnit
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D010767:Phosphorylcholine / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029681:Arabidopsis Proteins; D010455:Peptides; D010767:Phosphorylcholine