Search

Advances in mass spectrometry (MS) have enabled quick and accurate protein identification, an important analytical goal. However, knowledge of the identity of a protein only skims the surface of the structural and functional complexities that are characteristic of proteins. Described in this dissertation is the development of MS-based analytical techniques to investigate the structure of proteins using noncovalent interactions and photo-induced radical chemistry. The first half of the dissertation reports the discovery of a novel gas phase protein dissociation method using photo-induced radical chemistry to `direct' backbone fragmentation to specific amino acid residues, which we have called `radical directed dissociation' or RDD. Residue-specific dissociation, which is nearly unprecedented in the literature, is a significant advance towards enzyme-like protein disassembly in the gas phase. Investigation of numerous peptides and proteins yields two key features of RDD. First, fragmentation frequently occurs at tyrosine, phenylalanine, tryptophan, histidine, threonine, and serine. These residues have the lowest predicted C(beta)-H bond dissociation energies (BDEs) of the twenty canonical amino acids. Second, removal of the beta carbon of tyrosine abrogates backbone fragmentation at that residue. These results indicate that the C(beta) is the most important site for radical-induced backbone fragmentation and that radical migration in peptides appears to favor sites with the lowest C-H BDEs. We have called this latter phenomenon "radical funneling". Although useful for peptides, the "radical funneling" model becomes inappropriate for larger peptides and proteins, where structural effects cannot be ignored. Indeed, we show that radical migration is a sensitive reporter of tertiary structure of proteins in vacuo.The remaining half of the dissertation reports the development of selective noncovalent adduct protein probing mass spectrometry (SNAPP-MS) and radical migration

Advances in mass spectrometry (MS) have enabled quick and accurate protein identification, an important analytical goal. However, knowledge of the identity of a protein only skims the surface of the structural and functional complexities that are characteristic of proteins. Described in this dissertation is the development of MS-based analytical techniques to investigate the structure of proteins using noncovalent interactions and photo-induced radical chemistry. The first half of the dissertation reports the discovery of a novel gas phase protein dissociation method using photo-induced radical chemistry to `direct' backbone fragmentation to specific amino acid residues, which we have called `radical directed dissociation' or RDD. Residue-specific dissociation, which is nearly unprecedented in the literature, is a significant advance towards enzyme-like protein disassembly in the gas phase. Investigation of numerous peptides and proteins yields two key features of RDD. First, fragmentation frequently occurs at tyrosine, phenylalanine, tryptophan, histidine, threonine, and serine. These residues have the lowest predicted C(beta)-H bond dissociation energies (BDEs) of the twenty canonical amino acids. Second, removal of the beta carbon of tyrosine abrogates backbone fragmentation at that residue. These results indicate that the C(beta) is the most important site for radical-induced backbone fragmentation and that radical migration in peptides appears to favor sites with the lowest C-H BDEs. We have called this latter phenomenon "radical funneling". Although useful for peptides, the "radical funneling" model becomes inappropriate for larger peptides and proteins, where structural effects cannot be ignored. Indeed, we show that radical migration is a sensitive reporter of tertiary structure of proteins in vacuo.The remaining half of the dissertation reports the development of selective noncovalent adduct protein probing mass spectrometry (SNAPP-MS) and radical migration

Cytochrome P450 (CYP) family 4 constitutes monoxygenases responsible for hydroxylation of fatty acids and other lipids. For example, CYP4F3 metabolizes leukotrienes and CYP4F8 prostaglandin H. Importantly, six of the twelve CYP4 enzymes are orphans, i.e., with an unknown biological function. The catalytic activity of the enzyme CYP4F8 is known in seminal vesicles, but not in skin or psoriatic lesions, where CYP4F8 is highly expressed. The orphan CYP4F22 is also expressed in skin, and mutations in its gene has been linked to the rare skin disease lamellar ichthyosis, together with, inter alia, mutations in the genes of 12R-LOX and eLOX3. These enzymes appear to constitute a pathway producing hydroperoxides and epoxyalcohols from arachidonic acid. CYP4F22 is hypothesized to act in a consecutive step within this pathway. The aim of this thesis was to develop analytical methods to prepare and analyze hydroperoxides and epoxyalcohols derived from fatty acids by LC-MS/MS, and to investigate the catalytic performance of CYP4F8 and CYP4F22 for these substrates. The 12R-hydroperoxide of arachidonic acid (12R-HPETE) was prepared by autoxidation and separated from other hydroperoxides by chiral HPLC. MS/MS analysis showed that the hydroperoxides were unstable within the ion trap, but were stabilized by an increase in the isolation width. From the hydroperoxides, epoxyalcohols were generated by hematin treatment, and separated by normal phase HPLC. MS/MS spectra of several epoxyalcohols, derived both from arachidonic acid and linoleic acid, were characterized with aid of [2H]isotopomers and MS3 analysis. Apart from metabolic studies the thesis also include detailed information on MS/MS analysis of several oxygenated fatty acids, with proposed fragmentation mechanisms. The open reading frame of CYP4F22 was expressed in a recombinant yeast system, and LC-MS/MS analysis revealed that CYP4F22 catalyzed ω3 hydroxylation of arachidonic acid, but not any of the tested epoxyalcohols. I

Cytochrome P450 (CYP) family 4 constitutes monoxygenases responsible for hydroxylation of fatty acids and other lipids. For example, CYP4F3 metabolizes leukotrienes and CYP4F8 prostaglandin H. Importantly, six of the twelve CYP4 enzymes are orphans, i.e., with an unknown biological function. The catalytic activity of the enzyme CYP4F8 is known in seminal vesicles, but not in skin or psoriatic lesions, where CYP4F8 is highly expressed. The orphan CYP4F22 is also expressed in skin, and mutations in its gene has been linked to the rare skin disease lamellar ichthyosis, together with, inter alia, mutations in the genes of 12R-LOX and eLOX3. These enzymes appear to constitute a pathway producing hydroperoxides and epoxyalcohols from arachidonic acid. CYP4F22 is hypothesized to act in a consecutive step within this pathway. The aim of this thesis was to develop analytical methods to prepare and analyze hydroperoxides and epoxyalcohols derived from fatty acids by LC-MS/MS, and to investigate the catalytic performance of CYP4F8 and CYP4F22 for these substrates. The 12R-hydroperoxide of arachidonic acid (12R-HPETE) was prepared by autoxidation and separated from other hydroperoxides by chiral HPLC. MS/MS analysis showed that the hydroperoxides were unstable within the ion trap, but were stabilized by an increase in the isolation width. From the hydroperoxides, epoxyalcohols were generated by hematin treatment, and separated by normal phase HPLC. MS/MS spectra of several epoxyalcohols, derived both from arachidonic acid and linoleic acid, were characterized with aid of [2H]isotopomers and MS3 analysis. Apart from metabolic studies the thesis also include detailed information on MS/MS analysis of several oxygenated fatty acids, with proposed fragmentation mechanisms. The open reading frame of CYP4F22 was expressed in a recombinant yeast system, and LC-MS/MS analysis revealed that CYP4F22 catalyzed ω3 hydroxylation of arachidonic acid, but not any of the tested epoxyalcohols. I

We propose here a new strategy for the exhaustive mapping of phosphorylation sites in the Xenopus laevis Cdc25 phosphatase, which regulates cell cycle progression in eukaryotic cells. Two different MS analyses in a linear IT were used to identify the phosphorylated residues. First, a data‐dependent neutral loss (DDNL) analysis triggered the fragmentation of peptides that show enhanced neutral loss of phosphoric acid. Second, a targeted product ion scanning (TPIS) mass analysis was carried out in which MS2 events are triggered for specific m/z values. Full coverage of the protein sequence was obtained by combining the two analyses with two enzymatic digestions, trypsin and chymotrypsin, yielding a comprehensive map of the phosphorylation sites. Previous reports have shown Cdc25C to be phosphorylated by Cdc2–cyclin B at four residues (Thr48, Thr67, Thr138 and Ser205). By using this combination of scan modes, we have identified four additional phosphorylation sites (Thr86, Ser99, Thr112 and Ser163) in a recombinant Cdc25C protein containing 198 residues of the NH2‐terminal noncatalytic domain. The sensitivity of this combined approach makes it extremely useful for the comprehensive characterization of phosphorylation sites, virtually permitting complete coverage of the protein sequence with peptides within the mass detection range of the linear IT.

Mass spectrometry (MS) is a technique of paramount importance in Proteomics, and developments in this field have been possible owing to novel MS instrumentation, experimental strategies, and bioinformatics tools. Today it is possible to identify and determine relative expression levels of thousands of proteins in a biological system by MS analysis of peptides produced by proteolytic digestion. In some situations, however, the precise characterization of a particular peptide species in a very complex peptide mixture is needed. While single-fragment ion-based scanning modes such as selected ion reaction monitoring (SIRM) or consecutive reaction monitoring (CRM) may be highly sensitive, they do not produce MS/MS information and their actual specificity must be determined in advance, a prerequisite that is not usually met in a basic research context. In such cases, the MS detector may be programmed to perform continuous MS/MS spectra on the peptide ion of interest in order to obtain structural information. This selected MS/MS ion monitoring (SMIM) mode has a number of advantages that are fully exploited by MS detectors that, like the linear ion trap, are characterized by high scanning speeds. In this work, we show some applications of this technique in the context of biological studies. These results were obtained by selecting an appropriate combination of scans according to the purpose of each one of these research scenarios. They include highly specific identification of proteins present in low amounts, characterization and relative quantification of post-translational modifications such as phosphorylation and S-nitrosylation and species-specific peptide identification.

In this overview article celebrating the 25th anniversary of TrAC, we will discuss the progress of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem MS (LC-MS2) in environmental analysis. The article will cover challenges and achievements of LC-MS in environmental analysis with emphasis on the developments that occurred over 20 years and the impact caused by the discovery of electrospray and its applications to organic trace analysis. We will also outline the history of TrAC in relation to the publication of papers dealing with LC-MS applied to environmental analysis., The second part of the article will be devoted to LC-MS2 as the preferred method of choice in environmental analysis. We will report on applications on the use of triple quadupoles and hybrid technologies (e.g., Qq-LIT. Qq-TOF and Orbitrap) for a variety of the so-called emerging contaminants (e.g., pharmaceuticals, pesticides and endocrine disruptors).

We propose a method for deterministic implantation of single atoms into solids which relies on a linear ion trap as an ion source. Our approach allows a deterministic control of the number of implanted atoms and a spatial resolution of less than 1 nm. Furthermore, the method is expected to work for almost all chemical elements. The deterministic implantation of single phosphor or nitrogen atoms is interesting for the fabrication of scalable solid state quantum computers, in particular for silicon and diamond based schemes. A wide range of further applications is expected for the fabrication of nano and sub-nano electronic devices.

The feasibility of experimental design in combination with subsequent response surface modelling was illustrated for the prediction and interpretation of tandem mass spectrometric (MS/MS) fragmentation data using a linear quadrupole ion trap under various experimental conditions. The instrumental parameters included were (i) the pressure of the collision gas, (ii) the collision energy, (iii) the fill time of the linear ion trap and (iv) the scan rate. The spectral intensity and width of five fragment ions of the doubly charged neuro-active peptide bombesin were used for evaluation, and all experiments were performed so as to resemble the results obtained from a liquid chromatographic peak. The reported results show how fairly simple mathematical tools can be utilized sucessfully to describe fundamental mechanisms associated with multiple collisional activation and collision-induced dissociation processes without an extensively controlled experimental environment. Most beneficial, using the suggested approach, is the ability to study interaction (synergistic) effects between various parameters. As we realized from the results, many interaction effects are indeed significant. For example, the effect on the signal intensity of different collision gas pressure settings is strongly dependent on the settings of the other parameters. The described approach can easily be adopted for optimization purposes of any MS/MS experiment.

The feasibility of experimental design in combination with subsequent response surface modelling was illustrated for the prediction and interpretation of tandem mass spectrometric (MS/MS) fragmentation data using a linear quadrupole ion trap under various experimental conditions. The instrumental parameters included were (i) the pressure of the collision gas, (ii) the collision energy, (iii) the fill time of the linear ion trap and (iv) the scan rate. The spectral intensity and width of five fragment ions of the doubly charged neuro-active peptide bombesin were used for evaluation, and all experiments were performed so as to resemble the results obtained from a liquid chromatographic peak. The reported results show how fairly simple mathematical tools can be utilized successfully to describe fundamental mechanisms associated with multiple collisional activation and collision-induced dissociation processes without an extensively controlled experimental environment. Most beneficial, using the suggested approach, is the ability to study interaction (synergistic) effects between various parameters. As was realized from the results, many interaction effects are indeed significant. For example, the effect on the signal intensity of different collision gas pressure settings is strongly dependent on the settings of the other parameters. The described approach can easily be adopted for optimization purposes of any MS/MS experiment.