Analysis of Protein Phosphorylation in the Regions of Consecutive Serine/Threonine Residues by Negative Ion Electrospray Collision-Induced Dissociation. Approach to Pinpointing of Phosphorylation Sites

Abstract

Pinpointing of phosphorylation sites by positive ion collision-induced dissociation (CID) in phosphopeptides containing consecutive Ser/Thr residues (Ser/Thr clusters) is frequently hampered by the lack of backbone cleavage between adjacent Ser/Thr or pSer/pThr sites. In this study, we demonstrate that in negative ion collision-induced dissociation phosphorylated and unmodified residues of Ser/Thr clusters exhibit a very selective behavior toward cleavage of their N-Calpha bonds. Ser/Thr clusters were defined as two and more consecutive serine or threonine residues in phosphopeptide sequences. Dissociation reactions at pSer are significantly more abundant than those of unmodified sites. Thr residues exhibit the same effect, but the cleavages occurring at pThr are generally less prominent than those at pSer. The correlation observed between the facility of the amine backbone bond dissociation of phosphopeptides and the presence of the phosphate group on the side chain residues of Ser and Thr is attributed to the different magnitudes of electron density on the Calpha atoms of the amino acid in phosphorylated and unmodified forms. The results of this study indicate that the intensity ratio of the fragments generated by N-Calpha bond cleavage within the phosphopeptide Ser/Thr clusters represents a reliable and general marker for pinpointing of phosphorylation sites. The presented data illustrate that negative ion electrospray CID is superior over the standard positive ion mode approach for the localization of protein phosphorylation inside Ser/Thr clusters.

Solvatochromism and Nonradiative Decay of Intramolecular Charge‐Transfer Excited States: Bands‐of‐Energy Model, Thermodynamics, and Self‐Organization

“…There have been reports of the identification of phosphorylation by liquid chromatography/collision‐induced dissociation electrospray ionisation (LC/CID ESI) mass spectrometry (MS),5–8 negative ion atmospheric pressure ionisation (API) CID tandem mass spectrometry (MS/MS)9 and negative ion matrix‐assisted laser desorption/ionisation (MALDI) MS 10, 11. More recently, Lehmann and co‐workers have further described the use of negative ion ESI‐MS of phosphorylated Ser, Thr and Tyr residues (pSer, pThr and pTyr); pSer and pThr are identified by the process [(M‐H) − –H 3 PO 4 ] 12, 13…”

Application of the broadband collision‐induced dissociation (bbCID) mass spectrometry approach for protein glycosylation and phosphorylation analysis

“…Fragmentation of peptide anions is often governed by neutral loss reactions involving side chains rather than peptide backbone fragmentation. Therefore, the body of experimental information on MS/MS spectra of peptide anions is still much smaller than that for positive ions . However, our results suggest that the higher‐energy bbCID approach is useful for the assignment of phosphopeptides through the detection of phosphate diagnostic ions and H 3 PO 4 neutral losses.…”

Femtosecond laser‐induced ionization/dissociation tandem mass spectrometry (fsLID‐MS/MS) of deprotonated phosphopeptide anions

“…For a survey of a 20‐precursor data subset, a large proportion of c ions were observed to occur N‐terminal to either Ser or Thr (9/13 (69%) and 14/23 (61%) of c ‐ion occurrences for the 2‐ and 3‐precursors, respectively); the remainder of the c ions (and the number of their occurrences for the 2‐ and 3‐charge states combined) were: N‐terminal to pS (2), N‐terminal to D (2), N‐terminal to E (1), N‐terminal to L (1), and N‐terminal to the C‐terminal residue (7). From this data, it appears that c ‐ion formation favors backbone N–C ɑ cleavage adjacent to residues having low electron density at the C ɑ carbon; notably, such conditions have previously been reported as facilitating z ‐ion formation during negative‐mode CID‐MS/MS . Other diagnostic information is observable via 'x – 98' ions, which may be indicative of the phosphorylation site; an analysis of observable singly charged 'x–98' abundances originating from 20 [M–2H] 2– and 11 [M–3H] 3– precursors shows that the most abundant x n –98 ion (where 'n' is the phosphorylated residue) occurs in 13/20 instances (65%) and 6/11 instances (55%), respectively.…”