Effects of acidic peptide size and sequence on trivalent praseodymium adduction and electron transfer dissociation mass spectrometry

Abstract

Using the lanthanide ion praseodymium, Pr(III), metallated ion formation and electron transfer dissociation (ETD) were studied for 25 biological and model acidic peptides. For chain lengths of seven or more residues, even highly acidic peptides that can be difficult to protonate by electrospray ionization will metallate and undergo abundant ETD fragmentation. Peptides composed of predominantly acidic residues form only the deprotonated ion, [M + Pr − H]2+; this ion yields near complete ETD sequence coverage for larger peptides. Peptides with a mixture of acidic and neutral residues, generate [M + Pr]3+, which cleaves between every residue for many peptides. Acidic peptides that contain at least one residue with a basic side chain also produce the protonated ion, [M + Pr + H]4+; this ion undergoes the most extensive sequence coverage by ETD. Primarily metallated and non-metallated c- and z-ions form for all peptides investigated. Metal adducted product ions are only present when at least half of the peptide sequence can be incorporated into the ion; this suggests that the metal ion simultaneously attaches to more than one acidic site. The only site consistently lacking dissociation is at the N-terminal side of a proline residue. Increasing peptide chain length generates more backbone cleavage for metal-peptide complexes with the same charge state. For acidic peptides with the same length, increasing the precursor ion charge state from 2+ to 3+ also leads to more cleavage. The results of this study indicate that highly acidic peptides can be sequenced by ETD of complexes formed with Pr(III).

Electron transfer dissociation mass spectrometry of acidic phosphorylated peptides cationized with trivalent praseodymium

“…The location of the phosphorylated residue at the N‐terminus in pTyr Y‐8P may provide greater conformational flexibility in allowing the seven residues to coordinate with the Pr(III). Metal adduction consistently occurs for peptides with at least seven residues as observed in our previous studies . In the other phosphopeptide with an N‐terminal residue, pTyrL‐5P, the phosphorylated residue may be necessary to allow the peptide to coordinate with Pr(III).…”

“…This is in addition to the normal protonation sites,i.e., the N‐terminus and the side chains of arginine, lysine, and histidine. In the prior study, the 12 residue EGF(985‐996) with a proline formed the 4+ ion. However, EGF(988‐998) with a proline, a shorter chain (11 residues), and a highly acidic pY only forms [M + Pr] 3+ .…”

“…More uncertain is the formation of abundant [M + Pr] 3+ despite the highly acidic nature of all three peptides. In our previous work, a heptapeptide with six acidic residues only formed the deprotonated species [M + Pr – H] 2+ . The increase in chain length from seven to eight residues may contribute to the formation of [M + Pr] 3+ .…”

“…Here, the effect of Pr(III) adduction on ETD of acidic model and biological phosphorylated peptides is investigated. In two previous studies, we have observed abundant metallated peptide ion formation by ESI using Pr(III) for nonphosphorylated acidic peptides; these ions yield extensive sequence informative fragmentation by ETD. The current work also probes the effects of the location and identity of phosphorylated residue on ETD.…”

“…Although the use of ionization with metals is not yet routine in proteomic research, there is an increasing body of scientific work involving ECD and ETD of metallated peptides . Three studies have focused on metal cationization of phosphopeptides .…”