Phosphate group-driven fragmentation of multiply charged phosphopeptide anions. Improved recognition of peptides phosphorylated at serine, threonine, or tyrosine by negative ion electrospray tandem mass spectrometry.Anal Chem. 2006 Feb 15; 78(4):1249-56.AC
The nanoelectrospray product ion spectra of multiply charged phosphopeptide anions reveal the occurrence of phosphate-specific high-mass fragment ions of the type [M - nH - 79](n-1)-. These so far unrecognized fragments, which are observed for phosphoserine-, phosphothreonine-, and phosphotyrosine-containing peptides, are the counterparts of the established inorganic phosphopeptide marker ion found at m/z 79 = [PO3]-. The high-mass marker ions are formed with high efficiency at moderate collision offset values and are particularly useful for sensitive recognition of pSer-, pThr-, and pTyr-peptides due to the low background level in MS/MS spectra at m/z values above those of the precursor ions. By virtue of this feature, the detection of the new phosphorylation-specific fragment ions appears to be more sensitive than the detection of the low-mass phosphate marker ion at m/z 79, where a higher interference by nonspecific background signals is generally observed. The number of phosphate groups within a phosphopeptide can also be estimated on the basis of the [M - nH - 79](n-1)- ions, since these exhibit an effective, sequential neutral loss of H3PO4 of the residing phosphate groups. A mechanistic explanation for the formation of the [M - nH - 79](n-1)- ions from multiply charged phosphopeptides is given. The high-mass marker ions are proposed to originate from phosphopeptide anions, which carry two negative charges located at the phosphate group. A new search tool denominated "variable m/z gain analysis", which utilizes these newly recognized high-mass fragments for spotting of phosphopeptides in a negative ion parent scan, is proposed. The findings strengthen the value of negative ion ESI-MS/MS for analysis of protein phosphorylation.