Mass Spectrometry Based Covalent Ion/Ion Chemistry of Peptides and Proteins

Zhou Peng, Purdue University


Peptide and protein sequencing for the identification and structural characterization is a key application of tandem mass spectrometry in biological research. In an effort to influence fragmentation behavior or to facilitate spectral interpretation, many chemical derivatization strategies have been developed for the manipulation of proteins and peptides in solution phase. Gas-phase ion/ion reactions provide a means to manipulate ion type within the mass spectrometer, allowing rapid examination of the reaction products and facile comparison with the unmodified analytes. In this dissertation, gas-phase ion/ion chemistry involving new covalent reactions on peptide and protein ions are discussed. The first work demonstrates the reactivity of carboxylate moieties on peptide anions with N-hydroxysuccinimide (NHS) reagent cations to form anhydride bonds in the gas phase. The fragile anhydride bond is easily cleaved, resulting in an oxygen transfer from the carboxylate-containing species to the reagent, nominally observed as a water transfer. This work constitutes an example of distinct reactivities of a functional group between the gas and solution phases, and adds to the growing diversity of NHS chemistry in the gas phase in addition to the primary amine- and guanidine-specific reactions previously reported in our group. The carboxylic acid moieties on peptide cations are found to react with Woodward’s reagent K (wrk, N-ethyl-3-phenylisoxazolium-3’-sulfonate) anions to form a stable amide bond with loss of a neutral ketene derivative, leading to the addition of a mass tag on the C-terminus or aspartic acid/glutamic acid residues in peptides. Further MS/MS on the modified peptide ions results in a range of modified and unmodified b- and y- fragment ions, aiding the localization of the carboxylic acid functionalities. The conversion of carboxylic acids to ethyl amides also alters the fragmentation pattern of peptides, such as suppressing the aspartic acid effect. These findings not only explore fundamental gas-phase chemical reaction mechanisms, but also provide tools that aid the sequencing of peptides in bottom-up proteomics. Subsequently, wrk is expanded to a class of amidating reagents by a two-step solution-phase and gas-phase reactions. More interestingly, a C-terminal protected amino acid can also be conjugated to wrk as a reagent ion, which reacts with an ‘anchor’ peptide cation to realize gas-phase peptide extension at the C-terminus. This presents a novel method for peptide synthesis within the mass spectrometer that does not require the use of deprotecting agents, cleaving agents or solvent, rendering it a rapid and efficient method compared to the most common solid-phase peptide synthesis (SPPS) method nowadays. An ion/ion radical process allowing for the incorporation of selective cleavages in the structural characterization of peptide ions is described. A gas-phase approach is used for converting a sub-set of amino acid residues cations to dehydroalanine (Dha). An ion/ion reaction within the mass spectrometer between a multiply-protonated peptide and the sulfate radical anion introduces a radical site into the peptide reactant. Subsequent collisional activation of the peptide radical cation gives rise to radical side-chain loss from one of several particular amino acid side-chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z- ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. This gas-phase process for introducing selective cleavages in peptide cations constitutes a novel means for generating structural information in middle- and top-down tandem mass spectrometry.




MCLUCKEY, Purdue University.

Subject Area

Chemistry|Analytical chemistry

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