Large-Scale Examination of Factors Influencing Phosphopeptide Neutral Loss during Collision Induced Dissociation

Abstract

Collision-induced dissociation (CID) remains the predominant mass spectrometry based method for identifying phosphorylation sites in complex mixtures. Unfortunately, the gas-phase reactivity of phosphoester bonds results in MS/MS spectra dominated by phosphoric acid (H3PO4) neutral loss events, suppressing informative peptide backbone cleavages. To understand the major drivers of H3PO4 neutral loss, we performed robust non-parametric statistical analysis of local and distal sequence effects on the magnitude and variability of neutral loss, using a collection of over 35,000 unique phosphopeptide MS/MS spectra. In contrast to peptide amide dissociation pathways, which are strongly influenced by adjacent amino acid side chains, we find that neutral loss of H3PO4 is affected by both proximal and distal sites, most notably basic residues and the peptide N-terminal primary amine. Previous studies have suggested that protonated basic residues catalyze neutral loss through direct interactions with the phosphate. In contrast, we find that nearby basic groups decrease neutral loss regardless of mobility class, an effect only seen by stratifying spectra by charge-mobility. The most inhibitory bases are those immediately N-terminal to the phosphate, presumably due to steric hindrances in catalyzing neutral loss. Further evidence of steric effects is shown by the presence of proline which can dramatically reduce the presence of neutral loss when between the phosphate and a possible charge donor. In mobile proton spectra the N-terminus is the strongest predictor of high neutral loss, with proximity to the N-terminus essential for peptides to exhibit the highest levels of neutral loss.

Extensive non-canonical phosphorylation in human cells revealed using strong-anion exchange-mediated phosphoproteomics

“…It was noticeable that indeed, the majority (>70%) of phosphopeptides containing confidently localised pHis or pTyr (ptmRS>99%), were observed with no neutral loss, although this was not as marked for pSer/Thrcontaining peptides, where only 30% of peptides across all SAX fractions exhibited no neutral loss. These findings agree with previous observations demonstrating that phosphopeptide neutral loss propensity is dependent on both the type and relative position of amino acids within the sequence [42][43][44] . Further examination of the neutral loss ions (∆80 amu vs ∆98 amu vs ∆116 amu) revealed that the nature of the phosphorylated residue had very little effect on the likelihood of observation of a ∆80 amu ion from the precursor following HCD ( Fig.…”

Mechanistic insights into intramolecular phosphate group transfer during collision induced dissociation of phosphopeptides

“…10 On the other hand, the phosphopeptide systems studied by Cui and Reid exhibited high transfer ratios (approximately 50 times greater than average), 12 which beg the question what mechanism may be at play, and how general this mechanism may be. Surprisingly, the mechanistic study of phosphoric acid (H 3 PO 4 ) neutral loss 6,[15][16][17][18][19][20] has generated much more interest than has the mechanism for phosphosite scrambling. The only proposed mechanism for phosphate group transfer has cited the strong hydrogen bonding between the phosphate group and the protonated arginine side chain as a stabilizing factor allowing for the concerted transfer of the phosphate group and hydroxy hydrogen.…”

Mapping the Phosphorylation Pattern of Drosophila melanogaster RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry

“…Negative polarity peptide analysis generally results in uninformative CID mass spectra comprised mostly of neutral phosphate losses, precluding both identification and characterization of peptides. 12,13 Ultraviolet photodissociation (UVPD) using 193 nm photons is an alternative to existing collision- and electron-based activation methods that offers several advantages for phosphorylation site mapping in the CTD. Charge state bias, which is a limitation for traditional methods, is largely overcome using UVPD, and high sequence coverage has been demonstrated even for singly charged precursor ions.…”