<p>The combination of collision-activated dissociation, (CAD) and electron capture dissociation, (ECD) yielded a 125% increase in protein identification. The S-score was developed for measuring the information content in MS/MS spectra. This measure made it possible to single out good quality spectra that were not identified by a search engine. Poor quality MS/MS data was filtered out, streamlining the identification process.</p><p>A proteomics grade de novo sequencing approach was developed enabling to almost completely sequence 19% of all MS/MS data with 95% reliability in a typical proteomics experiment.</p><p>A new tool, Modificomb, for identifying all types of modifications in a fast, reliable way was developed. New types of modifications have been discovered and the extent of modifications in gel based proteomics turned out to be greater than expected.</p><p>PhosTShunter was developed for sensitive identification of all phosphorylated peptides in an MS/MS dataset.</p><p>Application of these programs to human milk samples led to identification of a previously unreported and potentially biologically important phosphorylation site.</p><p>Peptide fragmentation has been studied. It was shown emphatically on a dataset of 15.000 MS/MS spectra that CAD and ECD have different cleavage preferences with respect to the amino acid context.</p><p>Hydrogen rearrangement involving z• species has been investigated. Clear trends have been unveiled. This information elucidated the mechanism of hydrogen transfer.</p><p>Partial side-chain losses in ECD have been studied. The potential of these ions for reliably distinguishing Leu/Iso residues was shown. Partial sidechain losses occurring far away from the cleavage site have been detected. </p><p>A strong correlation was found between the propensities of amino acids towards peptide bond cleavage employing CAD and the propensity of amino acids to accept in solution backbone-backbone H-bonds and form stable motifs. This indicated that the same parameter governs formation of secondary structures in solution and directs fragmentation in peptide ions by CAD.</p>

The combination of collision-activated dissociation, (CAD) and electron capture dissociation, (ECD) yielded a 125% increase in protein identification. The S-score was developed for measuring the information content in MS/MS spectra. This measure made it possible to single out good quality spectra that were not identified by a search engine. Poor quality MS/MS data was filtered out, streamlining the identification process. A proteomics grade de novo sequencing approach was developed enabling to almost completely sequence 19% of all MS/MS data with 95% reliability in a typical proteomics experiment. A new tool, Modificomb, for identifying all types of modifications in a fast, reliable way was developed. New types of modifications have been discovered and the extent of modifications in gel based proteomics turned out to be greater than expected. PhosTShunter was developed for sensitive identification of all phosphorylated peptides in an MS/MS dataset. Application of these programs to human milk samples led to identification of a previously unreported and potentially biologically important phosphorylation site. Peptide fragmentation has been studied. It was shown emphatically on a dataset of 15.000 MS/MS spectra that CAD and ECD have different cleavage preferences with respect to the amino acid context. Hydrogen rearrangement involving z• species has been investigated. Clear trends have been unveiled. This information elucidated the mechanism of hydrogen transfer. Partial side-chain losses in ECD have been studied. The potential of these ions for reliably distinguishing Leu/Iso residues was shown. Partial sidechain losses occurring far away from the cleavage site have been detected. A strong correlation was found between the propensities of amino acids towards peptide bond cleavage employing CAD and the propensity of amino acids to accept in solution backbone-backbone H-bonds and form stable motifs. This indicated that the same parameter governs formation of secondary structures in solution and directs fragmentation in peptide ions by CAD.

Tandem mass spectrometry is widely used in proteomic studies because of its ability to identify large numbers of peptides from complex mixtures. In a typical LC-MS/MS experiment, thousands of tandem mass spectra will be collected and peptide identification algorithms are of great importance to translate them into peptide sequences. Though these spectra contain both m/z and intensity values, most popular protein identification algorithms primarily use predicted fragment m/z values to assign peptide sequences to fragmentation spectra. The intensity information is often undervalued, because it is not as easy to predict and incorporate into algorithms. Nevertheless, the use of intensity to assist peptide identification is an attractive prospect and can potentially improve the confidence of matches and generate more identifications. In this dissertation, an unsupervised statistical method, K-means clustering, was used to study peptide fragmentation patterns for both CID and ETD data, and many unique fragmentation features were discovered. For instance, strong c(n-1) ions were observed in ETD, indicating that the fragmentation site in ETD is highly related to the amino acid residue location. Based on the fragmentation patterns observed through data mining, a peptide identification algorithm that makes use of these patterns was developed. The program is named SQID and it is the first algorithm in our bioinformatics project. Our testing results using multiple public datasets indicated an improvement in the number of identified peptides compared with popular proteomics algorithms such as Sequest or X!Tandem. SQID was further extended to improve cross-linked peptide identification (SQID-XLink) as well as blind modification identification (SQID-Mod), and both of them showed significant improvement compared with existing methods. In this dissertation the SQID algorithm was also successfully applied to a mosquito proteomics project. We are incorporating new features and new algorithms to our software, such as more fragmentation methods, more accurate spectra prediction and more user-friendly interface. We hope the SQID project can continually benefit researchers and help to improve the data analysis of proteomics community.

Presented in this dissertation are FT-ICR H/D exchange and fragmentation studies of protonated peptides for the purpose of better understanding gas phase conformation and protonation motifs, and their affect on fragmentation patterns. In addition, a new ion activation method is developed to enhance the abundance of higher energy fragmentation pathways, thereby providing additional structural and/or mechanistic information.Studies to probe the relay mechanism of H/D exchange in small, model peptides found that residue position, proline configuration, and availability of the termini have a measurable effect on the H/D exchange behavior reflective of different conformation / protonation motifs.Charge remote cleavage C-terminal to aspartic acid was studied with the fixed charge derivative tris(2,4,6-trimethoxyphenyl) phosphonium (tTMP-P+). Ab initio calculations demonstrate that tTMP-P+ cannot activate the proposed aspartic acid nucleophile, the acidic side chain hydrogen initiates cleavage. Despite the absence of an ionizing proton, the derivatized peptide P+LDIFSDF (where P+ = tTMP-P+) exchanges three acidic hydrogens for deuterium. This supports the proposed aspartic acid cleavage mechanism that occurs without the direct involvement of an ionizing proton because acidic protons are able to participate in H/D exchange in the absence of an added proton. In addition, H/D exchange of P+LDIFSDF analogues provides insight into the sites and mechanisms of H/D exchange.H/D exchange to separate distinct peptide populations with differing rates and their subsequent fragmentation demonstrate that conformation / protonation motif affects the fragmentation spectrum observed. Studies of [P+LDIFSDF + H]2+, [RPPGFSPFR + 2H]2+ (bradykinin), and [RVYIFPF + 2H]2+ show that at least two distinct structures exist with different rates of H/D exchange and different fragmentation patterns. The overall MS/MS spectrum is a linear combination of all conformations and protonation motifs. In addition, fragmentation of labeled populations suggests that complementary bn+ / ym-n+ ion pairs are being formed from doubly-charged precursor by the same mechanism.SORI-RE CID is a new FT-ICR ion activation method based on the combination of sustained off-resonance irradiation (SORI) and on-resonant excitation (RE) with the purpose of enhancing higher energy fragmentation while maintaining low energy processes. The experiments presented serve to illustrate the usefulness of SORI-RE in diverse cases.

Bioinformatic methods were developed for improved identification of endogenous peptides using mass spectrometry. As a framework for these methods, a database for endogenous peptides, SwePep, was created. It was designed for storing information about endogenous peptides including tandem mass spectra. SwePep can be used for identification and validation of endogenous peptides by comparing experimentally derived masses of peptides and their fragments with information in the database. To improve automatic peptide identification of neuropeptides, targeted sequence collections that better mimic the peptidomic sample was derived from the SwePep database. Three sequence collections were created: SwePep precursors, SwePep peptides, and SwePep predicted. The searches for neuropeptides performed against these three sequence collections were compared with searches performed against the entire mouse proteome, and it was observed that three times as many peptides were identified with the targeted SwePep sequence collections. Applying the targeted SwePep sequence collections to identification of previously uncharacterized peptides yielded 27 novel potentially bioactive neuropeptides. Two fragmentations studies were performed using high mass accuracy tandem mass spectra of tryptic peptides. For this purpose, two databases were created: SwedCAD and SwedECD for CID and ECD tandem mass spectra, respectively. In the first study, fragmentation pattern of peptides with missed cleaved sites was studied using SwedCAD. It was observed that peptides with two arginines positioned next to each other have the same ability to immobilize two protons as peptides with two distant arginines. In the second study, SwedECD was used for studying small neutral losses from the reduced species in ECD fragmentation. The neutral losses were characterized with regard to their specificity and sensitivity to function as reporter ions for revealing the presence of specific amino acids in the peptide sequence. The results from these two studies can be used to improve identification of both tryptic and endogenous peptides. In summary, a collection of methods was developed that greatly improved the sensitivity of mass spectrometry peptide identification.

The surface-induced dissociation (SID) method is introduced into a Bruker matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI TOF MS) as an efficient ion fragmentation method. Ion trajectory calculations using the SIMION 7.0 ion optics program are performed and results are combined with simple unimolecular decay calculations in order to study the kinetics of the SID processes. In this instrument, the observation time frame for SID fragments lies in the submicrosecond region, allowing the specific detection of submicrosecond fragmentation channels. MALDI-produced protonated peptides in the mass range of 700 - 1500 Da and radical ions produced by laser irradiation of fullerenes C60 and C70 are fragmented at a gold surface coated with a self-assembled monolayer of alkanethiol to obtain TOF SID TOF mass spectra. For the SID of peptides in the hyperthermal energy regime, a fragmentation time frame of tens to a few hundreds of nanoseconds was calculated for the observed fast fragmentation channels (Chapters 3 and 4). Theoretical and experimental peak shape comparisons assuming unimolecular decay kinetics indicated a log rate constant in the range 6 - 7 (Chapter 4). Energy and mass resolved kinetic studies are also carried out. The contribution of special structural features to peptide fragmentation and the possibility of different fragmentation mechanisms such as sequential and parallel pathways are investigated. The results indicate a unimolecular decay process for observed fast peptide fragments ruling out a surface-shattering mechanism. Fullerene ions, especially C60+., showed a fragmentation behavior producing C2n+. fragments with an even number of C units at collision energies in the range of 100 - 400 eV (Chapter 5). At around 400 eV, additional small fragments appeared that are apart by only a single C unit. According to the calculated fragmentation times and the theoretical and experimental peak shape comparisons assuming unimolecular decay kinetics, both these processes may be approximated by parallel fast unimolecular decay processes with fragmentation time frames of tens to hundreds of nanoseconds although the poor theoretical and experimental peak shape matching for example in the decay of C60+. to C19+. may suggest deviations from a one-step unimolecular decay process.

No description available.

No description available.

Proteomic studies aim to identify, quantify and characterise the full complement of proteins in a cell or organism under a defined set of conditions, and are important to our understanding of cellular mechanisms. However, such studies represent a major analytical challenge. A typical proteome analysis involves enzyme-mediated digestion of complex protein mixtures to yield an even more complex mixture of peptides. Combined reverse-phase liquid chromatography and tandem mass spectrometry is then traditionally utilised to ascertain sequence information from the characteristic peptide sequences. Analytical data derived for the peptides are employed as search terms in database searching of protein sequences derived from gene sequences. The extreme complexity of the peptide mixtures analysed means that additional novel approaches are required to fully interrogate the vast number of tandem mass spectra generated, assigning peptide identity and thereby helping to address demanding biological questions. The research reported here aims to further our understanding of both gas phase peptide/peptide fragment ion structure and peptide fragmentation behaviour using a combination of tandem mass spectrometry and ion mobility measurement.To facilitate the determination of peptide ion collision cross section, a novel standard, QCAL-IM, produced using the QconCAT strategy, has been developed to enable calibration of drift time in Travelling Wave Ion Mobility instruments. The standard facilitates empirical determination of the rotationally averaged collision cross section of any peptide/peptide fragment ion that lies within the calibration range encompassed. QCAL-IM was subsequently utilised to determine the collision cross section of a range of peptide ions produced by Lys-C and Lys-N proteolysis of ‘standard’ proteins. Data produced allowed the effect upon gas phase ion conformation through changing the location of the basic residue lysine within a peptide sequence to be assessed.The fragmentation behaviour of peptide ions produced by a variety of digestion regimes during both collision-induced dissociation (CID) and electron transfer dissociation (ETD) has also been extensively studied. The proteases trypsin and Lys-C are those typically utilised during proteomic studies and peptides produced by each have either the basic residues arginine or lysine at their carboxy-terminus. Secondary enzymatic treatment with the exoprotease carboxypeptidase B cleaves these basic residues from the C-terminus. Tandem mass spectrometric analysis of both tryptic/Lys-C peptides and their CBPB truncated analogue highlights that the dominant fragment ion series observed during both CID and ETD is determined, at least in part, by the location of such basic residues.Finally, studies were undertaken to investigate the factors which may promote/inhibit scrambling of peptide fragment ion sequence, which has recently been shown to take place during CID. The effect of modifying the gas phase basicity of the N-terminal amino acid residue is studied through a combination of derivatisation and synthesis of alternative peptide sequences. Increasing the gas phase basicity is shown to inhibit the observed sequence scrambling while promoting concomitant rearrangement/retention of a carboxyl oxygen at the C-terminus to give enhanced formation of bn+H2O product ion species.

No description available.