<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>

In proteomic studies, separate experimental protocols have been necessary to identify proteins, determine their function, and predict their three-dimensional structure. In this study, a function-based separation of proteins was conceived to fractionate proteins prior to enzymatic digestion. In the initial demonstration of this technique, a DNA substrate was used to separate the DNA-binding proteins from the rest of the proteins in a lysate in order to identify protein function and to simplify the complex mixture of proteins. A total of 232 putative DNA-binding proteins and over 540 proteins in all were identified from E. coli. Hypothetical or unknown proteins were found, some of which bind to DNA. As a part of this demonstration, changes in protein expression caused by different environmental conditions (aerobic and anaerobic atmospheres) were observed. In a second demonstration, aimed at determining the three-dimensional structure of the DNA binding proteins, binding sites were blocked with oligonucleotides, and the modified proteins were purified, enzymatically digested, and subjected to tandem mass spectrometry. The amino acids in the DNA-binding domains of three proteins were determined. In a final application of function-based separation, DNA-binding proteins were digested with trypsin and the resulting peptides were separated using HPLC and subsequently analyzed using MALDI TOF/TOF and ESI Q-TOF instruments to study the complementary nature of the two ionization techniques, taking into account the differences between the mass analyzers. Based on the analysis of a large data set containing hundreds of peptides and thousands of individual amino acids, some of the currently held notions regarding the ionization processes were confirmed. ESI tends to favor the analysis of hydrophobic amino acids and peptides while MALDI is disposed toward mainly basic and aromatic species. These tendencies in ionization account in large part for the complementary nature of the peptides and proteins identified by the ESI and MALDI instruments and make it necessary to employ both types of instruments to gain the most information out of a given sample in a proteomics study. / Graduation date: 2004

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M.S.)--Rochester Institute of Technology, 2007. / Typescript. Includes bibliographical references (leaves 66-68).

Proteome profiling is the identification and quantitation of all proteins in biological samples. An important application of proteome profiling that has received much attention is clinical proteomics, a field that promises the discovery of biomarkers that will be useful for early diagnosis and prognosis of diseases. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from complex biological fluids and (ii) extensive biochemical processing (reduction, alkylation and enzymatic digestion) prior to analysis. However, the lack of standardized sample handling and processing in proteomics is a major limitation for the field. The conventional macroscale manual sample handling requires multiple containers and transfers, which often leads to sample loss and contamination. For clinical proteomics to be adopted as a gold standard for clinical measures, the issue of irreproducibility needs to be addressed. A potential solution to this problem is to form integrated systems for sample handling and processing, and in this dissertation, I describe my work towards realizing this goal using digital microfluidics (DMF). DMF is a technique characterized by the manipulation of discrete droplets (100 nL – 10 L) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. This thesis demonstrates how DMF can be a powerful tool capable of automating several protein handling and processing steps used in proteomics.

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.

The role played by the protein UnkG from the plant growth-promoting bacterium Pseudomonas putida UW4 in the ability of the bacterium to facilitate plant growth was studied. Previous work showed that over-expressing UnkG decreased the ability of P. putida UW4 to facilitate plant growth. In contrast, an unkG knock-out mutant of P. putida UW4 displayed an increased ability to promote plant growth. Various biological activities of P. putida UW4, P. putida UW4/pETP and P. putida UW4/pETP-unkG have been compared. Thus, the growth curves were measured; the Biolog™ system was used to test the ability of these strains to utilize various carbon sources; the strains were observed by scanning electron microscopy to assess their relative cell sizes; biochemical assays were conducted to quantify 3-indoleacetic acid production and to measure the enzymatic activity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase; proteome-level changes of P. putida UW4/pETP and P. putida UW4/pETP-unkG were profiled using two-dimensional difference in-gel electrophoresis (DIGE), followed by mass spectrometry identification of the altered proteins. After running DIGE, sixteen altered proteins were identified and their possible roles in the interactions between the bacterium and plants were discussed. Based on the preliminary results, we hypothesize that 1) UnkG may be detrimental to plant growth; 2) UnkG may negatively regulate a number of key cellular functions in a general way related to the energy balance of the bacterium.

Cerebrotendinous xanthomatosis (CTX), an autosomal recessive lipid-storage disorder with prominent neurological features, was first described by van Bogaert et al. in 1937. A deficiency of the mitochondrial sterol 27-hydroxylase due to mutations in the CYP27 gene (CYP27) blocks the oxidization of cholesterol side chain at the first step in the formation of bile acids. The accumulation of great amount of cholesterol and cholestanol in various tissues, especially in tendons and neural system, leads to the clinical symptoms including dementia, juvenile cataracts, xanthoma, cerebellar syndrome, atherosclerosis and a variety of neurological dysfunctions in CTX subjects. The diagnosis can be made by demonstrating elevated level of cholestanol in the serum and apprearance of xanthoma in tendon. There is a high prevalence of CTX in the Japanese, Sephardim Jewish and Italian populations. Here in this investigation, a one-reported pedigree of three affected individuals with typical characteristics of CTX and a heterozygous paternal carrier in Taiwan were assembled. The first part of the project was to clarify the genetic causes of these CTX patients and to design a series of analytical tests for achieving rapid and correct confirmation of the diagnosis. First, 3¡¦ and 5¡¦-flanking region as well as all 8 introns and 9 exons fragments of CYP27 were amplified from genomic DNA by polymerase chain reaction (PCR) and followed by single strand conformation polymorphism (SSCP) under optimal conditions. The SSCP patterns were identical among CTX subjects, the carrier, and normal controls for all exons except exon 2, implying some kind of mutation may exist on it. Then, direct DNA sequencer analysis was performed on the suspected PCR fragment of exon 2. A new homozygous mutation of one base-pair deletion of cytosine at codon 326 on exon 2 was found in all three CTX subjects in this family. This novel point deletion of cytosine at Pro102 (CCT) would cause a frameshift in mRNA (Pro102 ¡÷Leu) and result in the appearance of a premature termination condon (TGA) to substitute for Val106(GTG). This severe mistake would cause the breakdown in the normal function of sterol 27-hydroxylase and lead to CTX. However, gene analysis could not represent the corresponding functional proteins under various post-translational modifications in complex biological systems. Proteome is the set of expressed protein complement of a genome and proteomic analysis has been widely used in studies of life sciences. The second part of this study is to characterize the pathological mechanism of CTX patients with serum protein profiles and leukocytes isolated from CTX subjects by means of proteomic technologies, including two-dimensional electrophoresis (2-DE) and MALDI-TOF analysis. The results showed that the amount of vinculin, ABP-280, talin and vimentin in leukocytes of CTX patients increased significantly, reflecting the changes in membrane dynamics concerning cholestanol accumulation. The expression of target proteins in CTX patients and control was further confirmed by Western blotting with specific antisera which indicated the same tendency as 2-DE data. This is the first report to integrate both genomic and proteomic concepts for analyzing the possible mechanisms of CTX and information provided by this report should be very helpful for the future studies on CTX.

Thesis (MS)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Brian Bothner. Includes bibliographical references (leaves 67-80).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences