One-Step Peptide Backbone Dissociations in Negative-Ion Free Radical Initiated Peptide Sequencing Mass Spectrometry

Abstract

Peptide dissociation behavior in TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)-based FRIPS (free radical initiated peptide sequencing) mass spectrometry was analyzed in both positive- and negative-ion modes for a number of peptides including angiotensin II, kinetensin, glycoprotein IIb fragment (296-306), des-Pro(2)-bradykinin, and ubiquitin tryptic fragment (43-48). In the positive mode, the ·Bz-C(O)-peptide radical species was produced exclusively at the initial collisional activation of o-TEMPO-Bz-C(O)-peptides, and two consecutive applications of collisional activation were needed to observe peptide backbone fragments. In contrast, in the negative-ion mode, a single application of collisional activation to o-TEMPO-Bz-C(O)-peptides produced extensive peptide backbone fragmentations as well as ·Bz-C(O)-peptide radical species. This result indicates that the duty cycle in the TEMPO-based FRIPS mass spectrometry can be reduced by one-half in the negative-ion mode. In addition, the fragment ions observed in the negative-ion experiments were mainly of the a-, c-, x-, and z-types, indicating that radical-driven tandem mass spectrometry was mainly responsible for the TEMPO-based FRIPS even with a single application of collisional activation. Furthermore, the survival fraction analysis of o-TEMPO-Bz-C(O)-peptides was made as a function of the applied normalized collision energy (NCE). This helped us to better understand the differences in FRIPS behavior between the positive- and negative-ion modes in terms of dissociation energetics. The duty-cycle improvement made in the present study provides a cornerstone for future research aiming to achieve a single-step FRIPS in the positive-ion mode.

Photodissociation of TEMPO-modified peptides: new approaches to radical-directed dissociation of biomolecules

“…53 Advantageously, the TEMPO-based FRIPS methodology is also applicable to the study of negative ions. 29 Negative ion mass spectrometry provides structural information for proteomics that is both confirmatory and complementary to that obtained in the study of positive ions. Furthermore, for peptides with acidic residues or phosphorylation, negative ion mass spectrometry may be preferred for increased sensitivity.…”

“…Collisional activation of such radical ions results in side-chain and backbone cleavage with extensive sequence coverage. Lee et al introduced 2-[(2,2,6,6-tetramethylpiperidin-1-oxyl)methyl]benzoic acid (TEMPO-Bz, Scheme 1) as a free radical precursor bound to peptides through the N-terminus or at the ε-amine of lysine residues, [27][28][29] and is the tagging group employed in the present study. Collisional activation of the precursor peptide ions initiates homolytic cleavage of the labile NO-C bond, promoted by the remarkable stability of the released nitroxyl radical.…”

Experimental evidence for competitive N O and O C bond homolysis in gas-phase alkoxyamines

“…[46,68] The thermodynamic preference (ΔΔG 298 ) for O-C cleavage in deprotonated alkoxyamines in the present investigation is further enhanced by up to 20 kJ mol -1 compared to their neutral counterparts. This observation is true for all R 3 moieties studied herein, and thus trends in the effect of the R 3 moiety are representative of the trends in neutral species.…”

“…Only limited examples in the literature describe the tandem mass spectrometric analysis of alkoxyamines upon negative ion electrospray ionisation. [46] Commercially available nitroxides 4-carboxy-TEMPO…”

“…[38][39][40] It has previously been demonstrated that alkoxyamines undergo charge-remote homolysis of the O-C bond. [41][42][43] For example, Oh et al derivatized small peptides with alkoxyamines [44][45][46] and subjected these to electrospray ionisation (ESI) to form ions with the charge sites localised to amino acid residues and thus remote from the alkoxyamine. Subsequent collisioninduced dissociation (CID) of these ions resulted in almost exclusive O-C bond homolysis, generating an alkyl radical which initiated further fragmentation of the peptide ion.…”