Can collision‐induced negative‐ion fragmentations of [M–H]^– anions be used to identify phosphorylation sites in peptides?

Abstract

A joint experimental and theoretical investigation of the fragmentation behaviour of energised [M-H](-) anions from selected phosphorylated peptides has confirmed some of the most complex rearrangement processes yet to be reported for peptide negative ions. In particular: pSer and pThr (like pTyr) may transfer phosphate groups to C-terminal carboxyl anions and to the carboxyl anion side chains of Asp and Glu, and characteristic nucleophilic/cleavage reactions accompany or follow these rearrangements. pTyr may transfer phosphate to the side chains of Ser and Thr. The reverse reaction, namely transfer of a phosphate group from pSer or pThr to Tyr, is energetically unfavourable in comparison. pSer can transfer phosphate to a non-phosphorylated Ser. The non-rearranged [M-H](-) species yields more abundant product anions than its rearranged counterpart. If a peptide containing any or all of Ser, Thr and Tyr is not completely phosphorylated, negative-ion cleavages can determine the number of phosphated residues, and normally the positions of Ser, Thr and Tyr, but not which specific residues are phosphorylated. This is in accord with comments made earlier by Lehmann and coworkers.

Fragmentations of [M − H]− anions of peptides containing tyrosine sulfate. Does the sulfate group rearrange? A joint experimental and theoretical study

“…1), and GLGY(SO 3 H)VA(OH) ( À fragment anion is observed, [1] as are a series of characteristic a [1] and b prime [4] The two ions HOSO 3 À and [(M À H) À H 2 SO 4 ] À produce only minor peaks in the spectra of the four peptides under consideration, in marked contrast to major rearrangement peaks in the cognate negative ion spectra of peptides containing Tyr phosphate.…”

“…This loss of SO 3 is analogous to the loss of HOPO 2 from the [M À H] À anions of peptides containing Tyr(PO 3 H 2 ). [3,4] Scheme 2. Fig.…”

“…In contrast, the negative ion cleavages of tyrosine, serine, and threonine (Tyr, Ser and Thr) phosphates [3,4] are complex, involving a variety of S N i(P) rearrangements often followed by internal cyclization/cleavage reactions . [4] The phosphate group of Tyr can transfer to the Ser and Thr residues, and also to a carboxylate (or amide) moeity.…”

“…In summary, if a peptide containing any or all of Ser, Thr and Tyr is not completely phosphorylated, negative ion cleavages can determine the number of phosphorylated residues, and normally the positions of Ser, Thr, and Tyr, but not necessarily which specific residues are phosphorylated. [4] This paper investigates whether sulfate and phosphate groups behave similarly in the negative ion MS mode. The sulfate and phosphate groups are isobaric and both have large electron affinities making negative ion formation favorable.…”

“…[4] The phosphate group of Tyr can transfer to the Ser and Thr residues, and also to a carboxylate (or amide) moeity. In addition, a phosphate on Ser or Thr can transfer to a non-phosphorylated Ser or Thr (but not Tyr) or to a carboxylate group.…”