New Tandem Mass Spectrometric Methods of Structure and Sequence Elucidation of Synthetic and Tryptic Peptides
Viglino, Emilie Aude
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Over the last few decades, technological advances in the physical and life sciences have enabled scientists to look at various systems ever so intricately and to gather evidence for the mechanisms behind these systems, be they at the biological level or the astrophysical level. One of the many tools that has spurred the collection of large amounts of data for a better understanding of the World around us is mass spectrometry. Critical advancements in the engineering of mass spectrometers since their inception over a century ago have secured mass spectrometry’ position as a robust technology in the field of Chemistry. The versatility of this tool is illustrated by its application to a variety of scientific disciplines and by its predominance in the scientific literature. Currently, mass spectrometry is utilized routinely in the clinical laboratory as a means to evaluate the potential for certain biomarkers to be disease-causing. Such biomarkers typically are in protein or peptide form, hence the need to develop tools aimed at probing the sequences and structures of these compounds. Gaining insight into the building blocks of proteins and peptides and their overall organization is crucial for determining their functions and biological impact. The body of work presented herein focuses on the development of two separate tools for the study of peptides and proteins in the gas phase; the first tool was aimed at probing the structure of tyrosine-containing peptide cation radicals, which are particularly important in the functioning of redox enzymes, while the second tool was aimed at the synthesis of a unique iodine-containing charge tag in view of increasing the sequence coverage of digested proteins for easier and more reliable identification. In the first instance, a comprehensive study of collision-induced dissociation (CID) and near-UV photodissociation (UVPD) of a series of tyrosine-containing peptide cation radicals of the hydrogen-deficient and hydrogen-rich types is reported. Hydrogen-rich and hydrogen-deficient peptide cation radicals vary in electronic states and reactivity. Hydrogen-rich cation radicals [M + nH](n-1)+● are produced when an electron attaches to a multiply charged peptide ion, such as in electron transfer dissociation (ETD). Their backbone fragmentation is dominated by N-Cα cleavage and yields valuable sequencing information. Hydrogen-deficient cation radicals [M + (n-1)H]n+● are produced by an atom abstraction from a protonated peptide or via electron loss from a neutral peptide, as in electron ionization. Formation of the latter highly depends on the presence of both a basic amino acid and a tyrosine or tryptophan residue in the peptide and their fragmentation is dominated by C-Cα backbone cleavage. The former are far less studied and their mechanisms less understood. The generation and photodissociation of both hydrogen-rich and hydrogen-deficient cation radicals in tyrosine-containing pentapeptides was investigated in parallel studies. Using tandem analyses with an LTQ-XL ion-trap equipped with an ETD source and a λ=355nm Nd:YAG laser, crown-ether-peptide and Cu(II)(Terpy)-peptide complexes were formed via electrospray to generate [M+2H]+● and [M]+ ● cation radicals, respectively. Their subsequent photodissociation was achieved using a range of pulses, each conferring 18 mJ of energy to the cation radicals. The peptides investigated were AAAYR, AAYAR, AYAAR and YAAAR, with special interest in the effect of the tyrosine residue position. A validity experiment was conducted in order to mass-label the fragments obtained, where one alanine residue was substituted with one valine residue. All UVPD spectra obtained were compared to non-zero collision energy CID spectra. Additionally, the body of work described here reports the first application of UV/Vis photo- dissociation action spectroscopy for the structure elucidation of tyrosine peptide cation radicals produced by oxidative intra-molecular electron transfer in gas-phase metal complexes. Action spectroscopy exploits an optical parametric oscillator (OPO) for the tuning of a given laser frequency. In doing so, the dissociation channels for particular precursor ion are monitored as a function of wavelength, from λ = 200-700 nm in this case. For any intermediate species that may not be studied in solution, the action spectrum may be compared and correlated to putative ion structures obtained from density functional theory (DFT) calculations combined with time-dependent DFT (TD-DFT) calculations of electronic excitation energies and oscillator strengths in the cation radicals. The study showed that the oxidation of YAAAR produces Tyr-O radicals by combined electron and proton transfer involving the phenol and carboxyl groups. Oxidation of AAAYR produces a mixture of cation radicals involving electron abstraction from the tyrosine phenol ring and N- terminal amino group in combination with hydrogen-atom transfer from the Cα positions of the peptide backbone. In a second instance, a unique method of C-terminal lysine guanidination coupled to a diiodinated tyrosine insertion allowed the synthesis of a novel mass defect-containing charge tag. The potential for this diiodinated charge tag to enhance the electron transfer dissociation (ETD) efficiency of peptides and to thus increase their sequence coverage was evaluated for both synthetic and tryptic peptides. Iodine is a large atom with a large mass defect; this simply means that the nominal mass of iodine differs from its exact mass more so than is the case for the majority of the other atoms. The addition of iodine’s mass-defect is aimed at preventing the loss of information from low-mass cutoff typically seen in ion traps and also at shifting both precursor ions and their fragments to a spectral area with no native peptide fragments. Peptide fragmentation can generate several ion series, which results in complex spectra. Simplifying these spectra is one approach to enhancing peptide sequence coverage. Sequence identification commonly relies on the use of databases for spectral comparisons to known proteins. However, this approach suffers many shortcomings and has catalyzed the need for de novo sequencing, made possible by peptide tagging. Our mass defect labeling approach consisted in building the guanidine tag on a solid phase substrate bound to resin beads confined to a fritted syringe. Excess of thiocyanate-modified tyramine was then reacted with the substrate to form a thiourea analog before being oxidized and diiodinated to produce the precursor tag. In a last step, the tag is coupled with lysine-terminated peptides (synthetic or from digest) before being cleaved off the beads and analyzed. The mass spectral analyses of the tagged peptides products and their unmodified counterparts were conducted on at high-resolution. After optimization of the iodination process (3,5-diiodinated product yield >95%), lysine-terminated peptides were tagged. The diiodinated chargeable tag was successfully attached at the ɛ-amine of the lysine side chain of a mixture of synthetic lysine-terminated peptides (AAXAK, where X = D, F, H, K, N,). Overall, and across all five peptides, the ETD fragmentation efficiency was enhanced by the availability of multiple charges provided by the guanidine group and the complete coverage of the z-ion series, starting with z1, was made possible by addition of the iodinated charge tag. This method was applied to the peptides from bovine albumin (BSA) digests using Lys-C and Trypsin proteases in two separate experiments and showed equal success.
- Chemistry