Many mixtures important to research consist of hundreds or even thousands of individual components of interest. These types of mixtures are far too complex to separate by a single chromatographic dimension in any reasonable amount of time. However, if a multidimensional approach is used, where a complex mixture is separated by an initial dimension, simpler fractions of that separation are collected and each of those fractions are analyzed individually, highly complex mixtures can be resolved in relatively short amounts of time. This dissertation serves as a guide to multidimensional chromatography, in particular, two-dimension liquid chromatography. There are many aspects of multidimensional separations that have been investigated to show its aspects, drawbacks and potential ability to separate highly complex mixtures. Measurements for the performance of multidimensional chromatography, the effects of the first and subsequent dimensions and the approaches to pairing dimensions are shown with experimental examples. Fundamental and practical features of multidimensional chromatography are explained as well as theoretical discussions on current and future multidimensional chromatography performance. Experimentally, very high peak capacities were obtained (ca. 7000) and an algorithm to predict how to best optimize a two-dimensional separation based on the time used and performance was created for designing experiments.

Peptide sequencing by surface-induced dissociation (SID) on a MALDI-Ion Mobility-orthogonal-TOF mass spectrometer is demonstrated. The early version of the instrument used for proof-of-concept experiments achieves a mobility resolution of approximately 20 and TOF mass resolution better than 200. Peptide sequences of four peptides from a tryptic digest of cytochrome c (ca. 1 pmol deposited) were obtained. The advantage of IM-SID-o-TOFMS is that a single experiment can be used to simultaneously measure the molecular weights of the tryptic peptide fragments (peptide mass mapping) and partial sequence analysis, (real time tandem mass spectrometry.) Optimization of the MALDI-IM-SID-o-TOF mass spectrometer for peptide sequencing is discussed. SID spectra obtained by using stainless steel, Au grids, and fluorinated self-assembled monolayers (F-SAM) on Au are compared. Optimum collision energies differ for the various surfaces. The fragmentation patterns observed for a series of peptides and protein digests using the Nd:YAG laser (355 nm) for MALDI ion formation and an FSAM surface for ion activation is compared to the fragmentation patterns observed for CID and photodissociation. The fragmentation patterns observed in all cases are strikingly similar. Photodissociation produced a greater abundance of ions resulting from side-chain cleavages. As a general rule optimized SID spectra contain fewer immonium ions than either photodissociation or CID. Evaluation of an instrument incorporating a new hybrid drift cell is discussed. Spectra for a digest of hemoglobin is compared to that acquired with an ABI 4700 TOF-TOF. The performance of the instrument is also evaluated using a micro-crystal Nd:YAG laser (355 nm) for MALDI operated at 400 Hz. Experiments were performed to determine the sensitivity and overall performance of the instrument. The reproducibility of the MS/MS spectra for gramicidin S is shown to be 94% run-to-run. The best mobility resolution obtained for a neat deposition of the dye Crystal Violet was 60 t/∆t. Sensitivity was tested with the peptide fibrinopeptide A (m/z 1537, AA sequence ADSGEGDFLAEGGGVR). Data acquired for sixty seconds with approximately sixty femtomoles deposited. Abundant [M+H]+ ions where observed as well as [M+H]+-NH3 ions. The S/N for this short run was insufficient to identify any SID fragments

Work related musculoskeletal disorders are the dominating causes of reported ill-health in industrialized countries. These chronic pain conditions are one of the most costly public health problems in Europe and North America. When work related musculoskeletal disorders are considered to be of muscular origin and the trapezius muscle is affected, the common appellation is trapezius myalgia. Since little is known about the genesis or how it is maintained, it is of great importance to better understand the pathophysiology of trapezius myalgia; doing so will better enable recommendations for prevention, treatment and rehabilitation. Several hypotheses have been presented based on biochemical alterations in the muscle, suggesting increased signaling of inflammatory substances and altered metabolism. Previous research has not been able to present the comprehensive picture of the muscle in pain. Thus there is a demand for more comprehensive research regarding the biochemical milleu of the chronic trapezius muscle. Proteomic and metabolomic methods allow non-targeted simultaneous analyses of a large number of proteins and metabolites. The main emphasis in this thesis is on a proteomic method, two-dimensional differential gel electrophoresis (2D-DIGE). The method is validated to human skeletal muscle biopsy research with laboratory specific settings. In the baseline study, there were 14 metabolic, contractile, structural and regulatory proteins that differed significantly in abundance when trapezius and vastus lateralis muscles were compared. Using the validated 2D-DIGE method and the baseline study, a comparison between healthy and myalgic muscles was made. Biopsies from female cleaners with and without myalgia were compared to obtain results from women with the same type of work exposure. In the multivariate model, 28 identified unique proteins separated healthy and myalgic muscle and were grouped according to function: metabolic (n=10), contractile (n=9), regulatory (n=3), structural (n=4), and other (n=2). Finally, a second screening method, metabolomics, was introduced to analyze differences in metabolite content as a complement to and verification of the proteomic results. Gas chromatography-mass spectrometry (GC-MS) was performed on muscle interstitial fluid samples obtained with microdialysis, and differences in the abundance of extracellular metabolites were revealed.  The 2D-DIGE method is a reliable method to analyze human skeletal muscle. The outcomes of the proteomic analyses were dependant on the statistical approach. Systematic differences in protein and metabolite content were detected using a multivariate approach. Univariate analyses were used to analyze individual proteins for their significance. The significant proteins in the baseline study were predominately related to muscle fiber type which correlated with the differences in fiber type content between trapezius and vastus lateralis. The proteomic and metabolomics studies where myalgic and healthy muscles were compared provide us with new clues and new aspects regarding the pathophysiology of the myalgic muscle. Technically advanced methods employed in the thesis enabled an explorative screening of proteins of relevance for the pathophysiology of the myalgic muscle. The results of these analyses may contribute to the formulation of future hypothesis that need to be further evaluated.

The sequencing of genomes has caused a growing demand for functional analysis of gene products. This research field named proteomics is derived from the term proteome, which by analogy to genome is defined as all proteins expressed by a cell or a tissue. Proteomics is however methodologically restricted to the analysis of proteins with higher molecular weights. The development of a technology which includes peptides with low molecular weight and small proteins is needed, since peptides play a central role in many biological processes. To study endogenous peptides and hormones, the peptidome, an improved method comprising rapid deactivation in combination with nano-flow liquid chromatography (LC) and mass spectrometry (MS) was developed. The method has been used to investigate endogenous peptides in brains of mouse and rat. Several novel peptides have been discovered together with known neuropeptides. To elucidate the post mortem time influence on peptides and proteins, a time course study was performed using peptidomics and proteomics technologies. Already after three minutes a substantial amount of protein fragments emerged in the peptidomics study and some endogenous peptides were drastically reduced with increasing post mortem time. Of about 1500 proteins investigated, 53 were found to be significantly changed at 10 minutes post mortem as compared to control. Moreover, using western blot the level of MAPK phosphorylation was shown to decrease by 95% in the 10 minutes post mortem sample. A database, SwePep (a repository of endogenous peptides, hormones and small proteins), was constructed to facilitate identification using MS. The database also contains additional information concerning the peptides such as physical properties. A method for analysis of LC-MS data, including scanning for, and further profiling of, biologically significant peptides was developed. We show that peptides present in different amounts in groups of samples can be automatically detected. The peptidome approach was used to investigate levels of peptides in two animal models of Parkinson’s disease. PEP-19, was found to be significantly decreased in the striatum of MPTP lesioned parkinsonian mice. The localization and expression was further investigated by imaging MALDI MS and by in situ hybridization. The brain peptidome of reserpine treated mice was investigated and displayed a number of significantly altered peptides. This thesis demonstrates that the peptidomics approach allows for the study of complex biochemical processes.

In this study, I analyzed the Lactococcus lactis subspecies cremoris MG1363 stress response at sub-lethal alcohol levels during exponential growth phase at transcriptomics, proteomics, metabolomics levels. Ethanol, 1-butanol, 1-hexanol were the selected alcohols. Manganese- transporter- and arginine catabolic pathway genes were up-regulated by all alcohols suggesting they evoked oxidative and acidic stress. ATP manganese transporter genes, histidine- and galactose genes were also up-regulated. Purine- and pyrimidine synthesis genes were down-regulated. HPLC analysis displayed decreased biomass yield and glycolytic flux, suggesting increased glycolytic energy production and slowed down overall enzymatic rate. Proteomics analysis displayed differential expressed proteins associated with heat and oxidative stress.

Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes. The cleavage of ubiquitin chains from ubiquitinated proteins is performed by deubiquitinating enzymes, of which there are 16 in the Ubp (ubiquitin specific protease) group in the budding yeast Saccharomyces cerevisiae. The goal of my thesis has been to examine the biological roles and molecular functions of these enzymes using a combination of proteomic and molecular genetic approaches. As part of a large collaborative effort, interacting protein partners of the Ubps were isolated through affinity purification of tagged proteins, followed by protein identification by mass spectrometry. Purification of tagged Ubp6 led to the identification of the 19S proteasome complex, along with a novel subunit, Sem1. As the human homologue of Sem1 was previously identified as being associated with a protein involved in the repair of DNA double-strand breaks, I examined the possible role of Sem1 in DNA damage repair. A deletion of Sem1 and other 19S subunits resulted in hypersensitivity to various DNA damaging drugs, implicating the 19S complex in the process of DNA repair. iii I also found that purified Ubp2 interacted stably with the ubiquitin ligase Rsp5 and the protein Rup1. UBP2 interacts genetically with RSP5, indicating a functional relationship, while Rup1 facilitates the physical tethering of Ubp2 to Rsp5. Using the uracil permease Fur4, a Rsp5 substrate, as a model reporter, I found that ubp2Δ cells exhibited a temporal stabilization of Fur4 at the plasma membrane following the induction of endocytosis, implicating Ubp2 in protein sorting, specifically at the multivesicular body. In order to understand the role of Ubp2, I examined the effect of Ubp2 on Rsp5 function. I found that Rsp5, similar to its mammalian homologues, is auto-ubiquitinated in vivo, and that Ubp2 is able to directly deubiquitinate Rsp5 in vitro. Moreover, the presence of a substrate or Rup1 both resulted in increased autoubiquitination, implying an auto-inhibitory mechanism of Rsp5 regulation. Taken together, the data presented in this thesis implicate deubiquitinating enzymes in interesting and varied roles in the cell, and suggest a novel mechanism for the modulation of Rsp5-dependent trafficking processes.

Due to their simplicity, viruses require the assistance of host factors for various aspects of their replication cycle. This study investigated the interaction of one of the two non-structural replicase proteins of rubella virus (RUBV), P150, with cell proteins. RUBV forms replication complexes for replicating its RNA in association with membranes of endosomes and lysosomes; the thusly modified endosomes/lysosomes are termed cytopathic vacuoles or CPVs. In the first study, a RUBV expressing a FLAG epitope-tagged P150 was used to co-immunoprecipitate putative interacting cell proteins from an infected cell lysate fraction enriched for CPVs using differential centrifugation. However, the only interacting protein identified was the companion RUBV replicase protein P90. Thus, cell proteins do not bind with either sufficient affinity or in stoichiometric amounts to be detected by this method and may not be a component of the virus holoenzyme. In the second study, a proline-rich region within P150 with three PxxPxR consensus SH3 domain-binding motifs was investigated for its ability to bind cell proteins. Substitution mutations (to alanine) of the two prolines were made in each of these motifs with the finding that mutations in the first two motifs led to lower viral titers and a small plaque phenotype with reversion to the wt sequence within one passage. Mutations in the third motif had a wt phenotype and did not revert. However, these mutations did not affect viral RNA synthesis, suggesting that the importance of these motifs is in a later stage of viral life cycle, e.g. virion assembly and release. To extend these findings, the proline hinge region with either the wt or mutant sequence was expressed as a GST-fusion in human cells. Pulldown experiments revealed specific binding with human p32 protein (gC1qR), which was previously shown to interact with the RUBV capsid protein. Binding of p32 with P150 was confirmed. The function of p32 in the RUBV replication cycle is unclear, but could involve virion assembly and release or induction of apoptosis.

Ribosome-inactivating proteins (RIPs) such as Shiga-like toxin 1 (SLT-1) halt protein synthesis in eukaryotic cells by depurinating a single adenine base in the sarcin-ricin loop of 28S rRNA. The molecular details involved in the ER lumenal escape and subsequent site-specific depurination are lacking, despite a general understanding of the biochemical basis of SLT-1 toxicity. Using a combination of yeast-2-hybrid and HeLa lysate pull-down followed by LC-MS/MS we have discovered yeast and human proteins that interact with the catalytic A1 chain of SLT-1. Yeast-2-hybrid library screens followed by the expression of full-length protein candidates and pull-down experiments yielded Cue2 as the only yeast cellular component that binds to the SLT-1 A1 chain. Further truncational analysis revealed that the known protein domains (two Cue domains and a Smr domain) within the primary sequence of Cue 2 were not essential for the interaction. Cue2 is a yeast monoubiquitin binding protein of no known function that is structurally homologous to the human ubiquitin-associated domain which has been implicated in intracellular routing and ER-associated degradation. Pull-down experiments indicated that the mechanism by which the catalytic domain of RIPs cleaves its substrate involves initial docking interactions with the ribosomal stalk by virtue of a conserved acidic C-terminal peptide domain common to all three stalk proteins P0, P1, and P2. The A1 chain of SLT-1 transiently binds to this peptide with a modest binding constant and rapid on and off rates. Mutagenesis of charged residues within the A1 chain identified a cationic surface that interacts with the peptide motif. In addition, phage-display was used to rapidly probe the importance of each residue within this C-terminal ribosomal peptide. The analysis revealed a complementary acidic surface and an additional hydrophobic motif involved in the interaction. Moreover, deletion mutagenesis performed on the ribosomal protein P0 revealed that the A1 chain binds to an alternate site on P0 in proximity to the contact sites for P1/P2 heterodimers. These results demonstrate that the catalytic chain of RIPs such as SLT-1 dock on ribosomes using two classes of binding sites located within the ribosomal stalk which may aid in orienting their catalytic domain in close proximity to the depurination site.

Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes. The cleavage of ubiquitin chains from ubiquitinated proteins is performed by deubiquitinating enzymes, of which there are 16 in the Ubp (ubiquitin specific protease) group in the budding yeast Saccharomyces cerevisiae. The goal of my thesis has been to examine the biological roles and molecular functions of these enzymes using a combination of proteomic and molecular genetic approaches. As part of a large collaborative effort, interacting protein partners of the Ubps were isolated through affinity purification of tagged proteins, followed by protein identification by mass spectrometry. Purification of tagged Ubp6 led to the identification of the 19S proteasome complex, along with a novel subunit, Sem1. As the human homologue of Sem1 was previously identified as being associated with a protein involved in the repair of DNA double-strand breaks, I examined the possible role of Sem1 in DNA damage repair. A deletion of Sem1 and other 19S subunits resulted in hypersensitivity to various DNA damaging drugs, implicating the 19S complex in the process of DNA repair. iii I also found that purified Ubp2 interacted stably with the ubiquitin ligase Rsp5 and the protein Rup1. UBP2 interacts genetically with RSP5, indicating a functional relationship, while Rup1 facilitates the physical tethering of Ubp2 to Rsp5. Using the uracil permease Fur4, a Rsp5 substrate, as a model reporter, I found that ubp2Δ cells exhibited a temporal stabilization of Fur4 at the plasma membrane following the induction of endocytosis, implicating Ubp2 in protein sorting, specifically at the multivesicular body. In order to understand the role of Ubp2, I examined the effect of Ubp2 on Rsp5 function. I found that Rsp5, similar to its mammalian homologues, is auto-ubiquitinated in vivo, and that Ubp2 is able to directly deubiquitinate Rsp5 in vitro. Moreover, the presence of a substrate or Rup1 both resulted in increased autoubiquitination, implying an auto-inhibitory mechanism of Rsp5 regulation. Taken together, the data presented in this thesis implicate deubiquitinating enzymes in interesting and varied roles in the cell, and suggest a novel mechanism for the modulation of Rsp5-dependent trafficking processes.

Ribosome-inactivating proteins (RIPs) such as Shiga-like toxin 1 (SLT-1) halt protein synthesis in eukaryotic cells by depurinating a single adenine base in the sarcin-ricin loop of 28S rRNA. The molecular details involved in the ER lumenal escape and subsequent site-specific depurination are lacking, despite a general understanding of the biochemical basis of SLT-1 toxicity. Using a combination of yeast-2-hybrid and HeLa lysate pull-down followed by LC-MS/MS we have discovered yeast and human proteins that interact with the catalytic A1 chain of SLT-1. Yeast-2-hybrid library screens followed by the expression of full-length protein candidates and pull-down experiments yielded Cue2 as the only yeast cellular component that binds to the SLT-1 A1 chain. Further truncational analysis revealed that the known protein domains (two Cue domains and a Smr domain) within the primary sequence of Cue 2 were not essential for the interaction. Cue2 is a yeast monoubiquitin binding protein of no known function that is structurally homologous to the human ubiquitin-associated domain which has been implicated in intracellular routing and ER-associated degradation. Pull-down experiments indicated that the mechanism by which the catalytic domain of RIPs cleaves its substrate involves initial docking interactions with the ribosomal stalk by virtue of a conserved acidic C-terminal peptide domain common to all three stalk proteins P0, P1, and P2. The A1 chain of SLT-1 transiently binds to this peptide with a modest binding constant and rapid on and off rates. Mutagenesis of charged residues within the A1 chain identified a cationic surface that interacts with the peptide motif. In addition, phage-display was used to rapidly probe the importance of each residue within this C-terminal ribosomal peptide. The analysis revealed a complementary acidic surface and an additional hydrophobic motif involved in the interaction. Moreover, deletion mutagenesis performed on the ribosomal protein P0 revealed that the A1 chain binds to an alternate site on P0 in proximity to the contact sites for P1/P2 heterodimers. These results demonstrate that the catalytic chain of RIPs such as SLT-1 dock on ribosomes using two classes of binding sites located within the ribosomal stalk which may aid in orienting their catalytic domain in close proximity to the depurination site.