The number and types of diagnostic ions obtained by infrared multiphoton dissociation (IRMPD) and collision induced dissociation (CID) were evaluated for supercharged peptide ions created by electrospray ionization of solutions spiked with mnitrobenzyl alcohol. IRMPD of supercharged peptide ions increased the sequence coverage compared to that obtained by CID for all charge states investigated. Multiply charged, N-terminally derivatized peptides were subjected to electron transfer reactions to produce singly charged, radical species. Upon subsequent “soft” CID, highly abundant z-type ions were formed nearly exclusively, which yielded simplified fragmentation patterns amenable to de novo sequencing methods. Furthermore, the simplified series of z ions were shown to retain labile phosphoric acid moieties. Infrared multiphoton dissociation (IRMPD) was implemented in a novel dual pressure linear ion trap for rapid “top-down” proteomics. Due to secondary dissociation, IRMPD yielded product ions in significantly lower charge states as compared to CID, thus facilitating more accurate mass identification and streamlining product ion assignment. This outcome was especially useful for database searching of larger proteins (~29 kDa) as IRMPD substantially improved protein identification and scoring confidence. Also, IRMPD showed an increased selectivity towards backbone cleavages N-terminal to proline and C-terminal to acidic residues (especially for the lowest precursor charge states). Ultraviolet photodissociation (UVPD) at 193 nm was implemented on a linear ion trap mass spectrometer for high-throughput proteomic workflows. Upon irradiation by a single 5 ns laser pulse, efficient photodissociation of tryptic peptides was achieved with production of a, b, c, x, y, and z sequence ions, in addition to immonium ions and v and w side-chain loss ions. The factors that influence the UVPD mass spectra and subsequent in silico database searching via SEQUEST were evaluated. 193 nm ultraviolet photodissociation (UVPD) was employed to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a-/x-type, followed by d and w side-chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. LC-MS/UVPD analysis using high pH mobile phases yielded efficient characterization of acidic peptides from mitogen-activated protein kinases. / text

No description available.

No description available.

A derivatization method is applied to chemically modify the glucose molecules. Then the derivatized C1 and C2 labeled glucose can be differentiated by tandem mass spectrometry. A multiple reaction monitoring method is developed to quantify the C1- and C2-13C labeled glucose, with deuterated glucose as the internal standard.Based on the fragmentation of cross-linked amino acids (pyridinoline (PYD) and deoxypyridinoline (DPD)), a precursor ion scan method is developed to detect DPD and PYD from a complex matrix. DPD is detected in a hydrolyzed mouse ventricle collagen sample by this method.A series of peptides and proteins are successfully ionized by a home made DESI source. The investigation of the sample surface effect shows that self assembled monolayer surfaces produce better signal than bare gold surfaces, implying that this is due to the lower electron transfer on SAM film which allows more ions to survive.

The development of new technologies and the advancement of existing technical expertise can allow for dramatic improvements to be realized in analytical instrumentation. The development of an integrating solid-state ion detector, designed to have a high sensitivity as well as maintaining a high-level of stability, is described and evaluated. Several versions of the charge-transimpedance amplifier (CTIA) technology were constructed with different operating features. The CTIA-1 is a 32-pixel array detector designed for mass spectrometry. It has the capability to simultaneously detect multiple ion channels with a detection limit less than 100 ions. The CTIA-2 detector features an independent selectable gain for each detection channel. The CTIA-2 is a 4-channel device designed for ion mobility. Further design features were built into the CTIA-5 such as differential noise reduction capabilities.The CTIA-1 technology was evaluated for use in isotope ratio mass spectrometry on a custom-built Mattauch-Herzog mass spectrometer. An evaluation was conducted in terms of the detector sensitivity, stability, accuracy, precision, resolution, and mass bias. The CTIA-2 was tested on a sector mass spectrometer for its response to low ion currents of both positive and negative ions. The detector stability, its accuracy, and its precision were studied.The technique of ion mobility spectrometry is rapidly growing, as it is the main technology used for the detection of explosives at security checkpoints. The need to improve the sensitivity of existing ion mobility instruments has led to the exploration of using the CTIA detector in ion mobility instruments. Improvements in sensitivity of two to three orders of magnitude have been demonstrated using the described CTIA detectors. Additional applications that use ion mobility instruments for the detection of analytes have been presented, the chemical mapping of a halogen-contaminated sand bed, the detection of pesticides, as well as the detection of TNT in drinking water.Results indicate that the CTIA detector technology is well suited for use in both mass spectrometry and ion mobility. The sensitive and stable multi-array CTIA detectors perform well in isotope ratio mass spectrometry. Ion mobility instruments of all types can benefit from the added sensitivity supplied by this technology.

This thesis describes the recent improvements of the SMILETRAP Penning trap mass spectrometer and a number of interesting high precision mass measurements, which have been performed using the improved apparatus, and are relevant in todays fundamental physics problems. The mass of the hydrogen-like 24,26Mg ions as well as the masses of the hydrogen- and lithium-like 40Ca ions are presented in this work that are indispensable input values when evaluating g-factor measurements of the bound electron. In both cases the uncertainty in the masses was improved by one order of magnitude compared to the literature values known so far. The mass of 7Li has been measured and a new mass value has been obtained with an unprecedented relative uncertainty of 6.3x10-10. A large deviation of 1.1 μu (160ppb) compared to the literature value has been observed. In order to find the reason of this large deviation and to look for possible systematics we have measured the mass of 4He and 6Li and concluded that the 6Li(n,γ)7Li reaction Q-value used in the literature when calculating the 7Li mass is wrong by about 1 keV. The mass difference between 3He and 3H (Δ m (3H -3He)) is the Q-value of the tritium β-decay. An accurate knowledge of the tritium Q-value is of importance in the search for a finite rest mass of the electron neutrino. By adding a measurement of the mass of 3He1+ to previous mass measurement of 3H1+ and 3He2+ we have improved our previous Q-value by a factor of 2. At the moment our Q-value is the most accurate and more importantly it is based on the correct atomic mass values.

Formaldehyde cross-linking has been used to study protein-protein interactions in cells. Its short spacer arm, ability to permeate through cell membrane and the reversibility of the cross-linking reaction makes this a desirable cross-linker for in vivo studies. Although it has been widely used as a cross-linking reagent, the detailed chemistry behind protein cross-linking is not well understood. In vitro studies conducted under extended incubation periods (2 days) have shown that a multitude of amino acids are reactive to formaldehyde and that residue accessibility appears to play a role in reactivity. How applicable these findings are to formaldehyde cross-linking studies done under in vivo conditions (10-20 min incubations) is unclear. The chemistry of formaldehyde cross-linking was therefore investigated in model peptides under conditions similar to those used in in vivo studies. It was observed that only a subset of amino acids (amino termini and side chains of lysine and tryptophan) that were found reactive under extended incubation times was reactive in the much shorter incubation period. No cross-linking was detected between peptides, and elevating the peptide and formaldehyde concentrations resulted in only a minimal amount of cross-linked peptides. The relationship between residue accessibility and formaldehyde reactivity was assessed in model proteins that contain a more complex tertiary structure. It was shown that the extent of formaldehyde reactivity was dependent on the state of protein unfolding, i.e., solvent accessibility of reactive residues, and that an unfolded protein showed a significantly higher number of formaldehyde-induced modifications than a folded form, with lysine being the predominant reactive site. Formaldehyde treatment of proteins in their native form resulted in a low number of modifications even under an increased incubation time, suggesting that the protein remains folded during the course of the reaction. This is important for in vivo cross-linking studies where specificity and stability of protein-protein interactions is dictated by protein tertiary structure.

Contemporary proteomics studies require computational approaches to deal with both the complexity of the data generated, and with the volume of data produced. The amalgamation of mass spectrometry -- the analytical tool of choice in proteomics -- with the computational and statistical sciences is still recent, and several avenues of exploratory data analysis and statistical methodology remain relatively unexplored. The current study focuses on three broad analytical domains, and develops novel exploratory approaches and practical tools in each. Data transform approaches are the first explored. These methods re-frame data, allowing for the visualization and exploitation of features and trends that are not immediately evident. An exploratory approach making use of the correlation transform is developed, and is used to identify mass-shift signals in mass spectra. This approach is used to identify and map post-translational modifications on individual peptides, and to identify SILAC modification-containing spectra in a full-scale proteomic analysis. Secondly, matrix decomposition and projection approaches are explored; these use an eigen-decomposition to extract general trends from groups of related spectra. A data visualization approach is demonstrated using these techniques, capable of visualizing trends in large numbers of complex spectra, and a data compression and feature extraction technique is developed suitable for use in spectral modeling. Finally, a general machine learning approach is developed based on conditional random fields (CRFs). These models are capable of dealing with arbitrary sequence modeling tasks, similar to hidden Markov models (HMMs), but are far more robust to interdependent observational features, and do not require limiting independence assumptions to remain tractable. The theory behind this approach is developed, and a simple machine learning fragmentation model is developed to test the hypothesis that reproducible sequence-specific intensity ratios are present within the distribution of fragment ions originating from a common peptide bond breakage. After training, the model shows very good performance associating peptide sequences and fragment ion intensity information, lending strong support to the hypothesis.

En la presente Tesis Doctoral se han desarrollado diferentes métodos analíticos para la determinación de pharmaceuticals and personal care products (PPCPs) en aguas medioambientales. El término PPCPs engloba todos los fármacos, drogas de abuso, hormonas y los excipientes activos incluidos en productos de uso personal, así como los metabolitos, conjugados y sub-productos de transformación. Los métodos desarrollados han estado basados en extracción en fase sólida o extracción con barras magnéticas agitadoras seguidos de la cromatografía líquida acoplada a la espectrometría de masas o de masas en tándem. Debido a los bajos límites de detección alcanzados, estos métodos se pudieron aplicar a la monitorización de estos compuestos en diferentes plantas depuradoras del área de Tarragona, donde la información al respeto era inexistente. Debido a que el principal destino de las aguas residuales son los ríos, se analizaron aguas del Ter, Llobregat y Ebro. Para completar el estudio, la presencia de PPCPs fue determinada en aguas de consumo. / In the framework of the present Doctoral Thesis different analytical methods have been developed to determine pharmaceuticals and personal care products (PPCPs) in environmental waters. The term PPCPs cover all the pharmaceuticals, drugs of abuse, hormones, the active compounds included in personal care products, and also metabolites, conjugates and transformation sub-products. The developed methods were based on solid-phase extraction and stir bar sorptive extraction followed by liquid chromatography coupled to mass spectrometry and tandem mass spectrometry. Achieving the low limits of detection, these methods could be applied to the monitoring of these compounds in different sewage treatment plants from Tarragona Region, where not data were available. The PPCPs resulting in the effluent sewage waters can achieve rivers. Therefore, waters from three rivers: Ter, Llobregat and Ebro were analyzed. To complete the study, also drinking water was analyzed to determine PPCPs.

One of the best ways to diagnose breast cancer early or to predict therapeutic response is to use serum biomarkers. Unfortunately, for breast cancer, we do not have effective serological biomarkers. We hypothesized that novel candidate tumor markers for breast cancer may be secreted or shed proteins that can be detected in tissue culture supernatants of human breast cancer cell lines. A two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) strategy was utilized to identify and compare levels of extracellular and membrane-bound proteins in the conditioned media. Proteomic analysis of the media identified in excess of 600, 500 and 700 proteins in MCF-10A, BT474 and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6 and 10 (ranging in concentration from 2-50 µg/L), as validated by ELISA and confidently identified HER-2/neu in BT474 cells. Sub-cellular localization was determined based on Genome Ontology (GO) for the 1,139 proteins, of which 34% were classified as extracellular and membrane-bound. Tissue specificity, functional classifications and label-free quantification were performed. The levels of eleven promising molecules were measured in biological samples to determine its discriminatory ability for control versus cases. This screen yielded activated leukocyte cell adhesion molecule (ALCAM) as a promising candidate. The levels of ALCAM, in addition to the classical breast cancer tumor markers carbohydrate antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) were examined in 300 serum samples by quantitative ELISA. All three biomarkers effectively separated cancer from non-cancer groups. ALCAM, with area under the curve (AUC) of 0.78 [95% CI: 0.73, 0.84] outperformed CA15-3 (AUC= 0.70 [95% CI: 0.64, 0.76]) and CEA (AUC= 0.63 [95% CI: 0.56, 0.70]). The incremental values of AUC for ALCAM over that for CA15-3 were statistically significant (Delong test, p <0.05). Serum ALCAM appears to be a new biomarker for breast cancer and may have value for disease diagnosis.