Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

Abstract

Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno) phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se–S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se–S cleavage, analogous to the S–S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se–S of the tag to the S–S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se–S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.

Characterization of the Cysteine Content in Proteins Utilizing Cysteine Selenylation with 266 nm Ultraviolet Photodissociation (UVPD)

“…It was previously reported that NPSP-derivatized peptides undergo Se-S cleavage upon ETD or CID [28]. Thus, for comparative purposes, collision-based (CID and HCD) and ETD promoted cleavage of up to two S-Se cleavages in conjunction with charge reduction for tagged aprotinin; the analogous MS/MS spectra for cytochrome c were not readily interpretable.…”

“…Fast, efficient, and selective selenylation of cysteine-containing species has been achieved previously using NPSP (Scheme 1) [27][28][29]. Additionally, online electrolytic cleavage of disulfides via an electrochemical cell prior to reactions with NPSP and mass spectrometry analysis has been reported as a means to identify disulfide-containing peptides in digests [30].…”

Cysteine-Selective Peptide Identification: Selenium-Based Chromophore for Selective S–Se Bond Cleavage with 266 nm Ultraviolet Photodissociation

“…NPSP has been shown previously to yield fast and efficient derivatization of free cysteine residues. [52][53][54][55] Other methods for detecting cysteine-containing residues based on mass shifts due to the loss of different hydroquinone derivatives have also been explored and shown to …”

Selective cleavage upon ETD of peptides containing disulfide or nitrogen–nitrogen bonds

“…Several groups have begun to explore the derivatization of peptides with tags containing selectively cleavable groups in order to promote bond-selective cleavages [29][30][31][32][33][34][35][36]. Part of this strategy is derived from the concept that low energy collisional activation favors cleavage of the most labile bonds, thus motivating interest in installing bonds with low dissociation energies to enhance site-specific cleavages.…”

“…Typical energies of CÀ ÀC and CÀ ÀO bonds fall in the range of 350-360 kJ/mol [37], whereas others are much lower: an SÀ ÀS bond energy is around 305 kJ/mol, a CÀ ÀS bond energy is around 270 kJ/mol, and an NÀ ÀN bond falls in the range of 165 kJ/mol, less than half of the energy of CÀ ÀC and CÀ ÀO bonds [37]. One group reported the cleavage of SeÀ ÀS bonds in protonated peptides upon CID or ETD, in which the unusual SeÀ ÀS bond was installed via the derivatization of cysteine-containing peptides with an appropriate seleniumcontaining reagent [36]. Other groups have incorporated tags, such as TEMPO [34], Vazo 68 [33], or N-terminal lysine side chain derivatization to t-butyl peroxycarbamate, that generate radical ions upon CID [29].…”