Structures Derived from Mass Spectrometric Peptide Fragmentation Pathways Explored by Combined Experimental and Theoretical Approaches
Final Report Abstract
High-throughput protein identification and quantification in proteomics are primarily based on sequencing of proteolytic peptides by means of tandem mass spectrometry (MS/MS). In these sequencing experiments peptides are ionized by protonation then introduced into the mass spectrometer where they undergo fragmentation following collisions with inert gas atoms/molecules (collision-induced dissociation (CID)). The resulting spectra which often contain abundant sequence informative b and y fragments, are used to deduce the peptide sequences normally via the use of bioinformatics tools. These programs utilize fragmentation models to generate theoretical spectra for candidate sequences then measure the similarity between these theoretical spectra and the experimental MS/MS spectra. This strategy only performs well if the fragmentation model implemented predicts MS/MS spectra accurately. Regrettably, this is often not the case, so current sequencing software works with a high level of false positive identifications. Utilizing more realistic fragmentation models should considerably improve the bioinformatics tools and therefore the accuracy of identifications of peptide sequence. Consequently, significant research effort is currently devoted to understanding the underlying peptide fragmentation chemistry and the gas-phase structures responsible for it. In this project we used state-of-the-art experimental and theoretical methods to study the structure and reactivity of gas-phase peptide ions and their fragments. The applied experimental techniques involved tandem mass spectrometry, 'action' IR spectroscopy, stabile isotope labeling, and ion mobility spectrometry (IMS). The 'raw' data provided by these methods were processed with the help of theory using modeling and density functional theory calculations. IR spectroscopy of gas-phase peptide ions and fragments combined with theory produced invaluable insights into the structure and dissociation chemistry of these species. Our studies indicated that K+ tagged bradykinin fragment 1-5 forms a zwitter-ionic structure in the gas phase while K+ tagged leu-enkephalin exists as a charge-solvated species. IR studies indicated that leu-enkephalin is protonated at the N-terminal amino group and protonated Ala n oligopeptides feature different conformation motifs depending on the size of the peptide. Our IR, IMS, and theoretical studies demonstrated that the Ö fragment of protonated leu-enkephalin is terminated by an oxazolone ring at the C-terminus. Similar studies on the 34 fragment demonstrated the co-existence of linear and 4 macro-cyclic isomers. Time-dependent IR studies on the b4 fragment provided the first direct evidence for the mobile proton that is transferred between two competing protonation sites. We have demonstrated hat CID of protonated peptides can lead to abundant nondirect sequence fragment ions that cannot directly be derived from the primary peptide structure. Experimental and theoretical evidence indicated that the primary b fragments can undergo a major rearrangement where the carbon center of the oxazolone ring is attacked by the N-terminal amino group to induce formation of cyclic peptide b isomers. The latter can undergo various proton transfer reactions and open up to form other than the original linear b5 isomer leading to scrambling of sequence information in CID of protonated peptides. Elimination of ammonia from a fragments involves another rearrangement replacing the formal C-terminal residue to the N-terminus. Energetic, mechanistic, and kinetic details of these chemistries were determined in our combined experimental and theoretical studies.
Publications
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Infrared Fingerprint Spectroscopy and Theoretical Studies of Potassium Ion Tagged Amino Acids and Peptides in the Gas Phase, J. Am. Chem. Soc, 2005, 127, 8571-8579
N. C. Polfer, B. Paizs, L. C. Snoek, I. Compagnon, S. Suhai, G. Meijer, G. von Helden and J. Oomens
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Spectroscopic and Theoretical Evidence for Oxazolone Ring Formation in Collision Induced Dissociation of Peptides, J. Am. Chem. Soc, 2005, 127, 17154-17155
N. C. Polfer, J. Oomens, S. Suhai and B. Paizs
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Isotope Labeling and Theoretical Study of the Formation of a3* lons from Protonated Tetraglycine, J. Am. Soc Mass Spectrom., 2006, 17, 1654-1664
T. Cooper, E. Talaty, J. Grove, M. Van Stipdonk, S. Suhai, and B. Paizs
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Revising the Proton Affinity Scale of the Naturally Occurring a-amino Acids, J. Am. Soc. Mass Spectrom., 2006, 17, 1275- 1281
C. Bleiholder, S. Suhai and B. Paizs
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Scrambling of sequence information in collision-induced dissociation of peptides, J. Am. Chem. Soc, 2006, 128, 10364-10365
A. G. Harrison, A. B. Young, C. Bleiholder, S. Suhai and B. Paizs
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Backbone Cleavages and Sequential Loss of Carbon Monoxide and Ammonia from Protonated AGG: A Combined Tandem Mass Spectrometry, Isotope Labeling, and Theoretical Study, J. Am. Soc. Mass Spectrom., 2007, 18, 1291-1303
B. J. Bythell, D. F. Barofsky, F. Pingitore. C. Wesdemiotis, and B. Paizs
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Infrared Spectroscopy and Theoretical Studies on Gas-phase Protonated Leu-enkephalin and Its Fragments: Direct Experimental Evidence for the Mobile Proton, J. Am. Chem. Soc, 2007, 129, 5887-5897
N. C. Polfer, J. Oomens, S. Suhai and B. Paizs
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On the Dynamics of Fragment Isomerization in Collision-Induced Dissociation of Peptides, J. Phys. Chem. A, 2008,112, 1286-1293
N. C. Polfer, B. C. Bohrer, M. D. Plasencia, B. Paizs, and D. E. Clemmer
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Sequence Scrambling Fragmentation Pathways of Protonated Peptides, J. Am. Chem. Soc, 2008, 130, 17774-17789
C. Bleiholder, S. Osburn, T. D. Williams, S. Suhai, M. Van Stipdonk, A. G. Harrison, and B. Paizs
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Structure and Reactivity of an and an* Peptide Fragments Investigated Using Isotope Labeling, Tandem Mass Spectrometry, and Density Functional Theory Calculations, J. Am. Soc. Mass Spectrom., 2008, 19, 1788-1798
B. J. Bythell, S. Molesworth, S. Osburn, T. Cooper, B. Paizs, M. Van Stipdonk
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Vibrational Spectroscopy and Conformational Structure of Protonated Polyalanine Peptides Isolated in the Gas Phase, J. Phys. Chem. A, 2008. 112, 4608- 4616
T. D. Vaden, T. S. J. A. de Boer, J. P. Simons, L. C. Snoek, S. Suhai, and B. Paizs,
