Protein derivatization and sequential ion/ion reactions to enhance sequence coverage produced by electron transfer dissociation mass spectrometry

Abstract

Previously, we described implementation of a front-end ETD (electron transfer dissociation) source for an Orbitrap instrument (1). This source facilitates multiple fills of the C-trap with product ions from ETD of intact proteins prior to mass analysis. The result is a dramatic enhancement of the observed ion current without the need for time consuming averaging of data from multiple mass measurements. Here we show that ion-ion proton transfer (IIPT) reactions can be used to simplify ETD spectra and to disperse fragment ions over the entire mass range in a controlled manner. We also show that protein derivatization can be employed to selectively enhance the sequence information observed at the N- and C-termini of a protein.

Keywords: ETD; IIPT; protein derivatization.

Figure 1. ETD/IIPT MS/MS spectra recorded on intact apomyoglobin

(A) ETD spectrum recorded on (M+26H)²⁶⁺ ions from apomyoglobin using a reaction time of 5 msec. (B–E) Spectra obtained by performing IIPT reactions on the ETD fragment ions in (A) for 20 msec, 40 msec, 80 msec, and 160 msec, respectively.

Figure 2. ETD reaction mechanism

Fragmentation scheme for the production of ions of types c and z by cleavage of the N-Cα bond of a multiply charged precursor following the transfer of an electron from a radical anion of fluoranthene.

Figure 3. Reaction of sulfur hexafluoride anion with a positively charged ETD fragment ion

The sulfur hexafluoride radical anion removes a proton from the fragment, cⁿ⁺, reduces charge on the fragment by one and forms sulfur pentafluoride plus HF.

Figure 4. Fragment ions observed with long and short IIPT reaction times

MS/MS spectra recorded on (M+26H)²⁶⁺ ions from apomyoglobin using an ETD reaction time of 5 msec and (A) an IIPT reaction time of 160 msec or (B) an IIPT reaction time of 20 msec.

Figure 5. Heat Map of the sequence coverage generated from intact apomyoglobin by using sequential ETD/IIPT reactions

Fragment ions observed with 5 msec of ETD and 160 msec of IIPT are lightly shaded and dominated by charge states of +1 and +2. More highly charged fragment ions (+3 to +7) that are observed for the first time at shorter IIPT reaction times (80, 40, and 20 msec) are labeled in darker shades. Those shaded in black were only observed following chemical derivatization of the protein.

Figure 6. Derivatization of human CLIP peptide with histamine enhances fragmentation observed by ETD

(A) ETD MS/MS spectrum of [M+3H]⁺³ ions m/z 822.91) from unmodified CLIP. (B) ETD MS/MS spectrum of [M+7H]⁺⁷ ions (m/z 433.27) of CLIP derivatized with histamine. Following derivatization, carboxyl groups of five, D and E residues, plus the C-terminus are converted to amides containing a basic imidazole ring. (C) Sequence coverage obtained from (B). Residues labeled by a histamine group are indicated by an asterisk.

Figure 7. Protein modification strategies for observing extreme N- and C-terminal fragment ions and for enhancing fragmentation by ETD

(A) Derivatization of the C-terminal carboxyl group (and other acidic residues) with aminoethylbenzimidazole. (B) Derivatization of the amino terminus (and other side chain amino groups) with a protected thiopropionic acid hydroxysuccinimide ester followed by reaction with aminoethylmaleimide.

Figure 8. Sequence coverage of the N- and C-terminus of apomyoglobin following derivatization

(A) 200–1700 m/z subsection of an ETD/IIPT MS/MS spectrum of apomyoglobin derivatized with aminoethylbenzimidazole. Observed z-ions are indicated. (B) 250–1000 m/z subsection of an ETD/IIPT MS/MS spectrum of apomyoglobin derivatized with DTSSP-maleimide. Observed c- and z-ions are indicated. Labeled amino acid residues are indicated by an asterisk. ¥ denotes fragment ions not carrying a covalent label.