Reporter Ion Characterization for Protein Quantification Studies Based on Isobaric Tags. Protein quantification is an important aspect of proteome characterization and is crucial in understanding biological mechanisms and human diseases. Discovery-based or un-targeted studies have often used covalent tagging strategies (i.e., iTRAQ, TMT) where reporter ion signals collected in the tandem MS experiment are used for the quantification. However, it has been difficult to establish the relative changes in reporter ion signals that are required to detect significant changes at the protein level. In our studies, the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation was evaluated. In order to better understand the behavior of the reporter ions we have evaluated the use of within spectra normalization, that we have termed row-normalization. When applied to replicated protein mixtures of equal concentration, the distribution of ion ratios were found to have a normal Gaussian distribution, and the width of the distribution can be used to establish confidence levels for a given reporter ion ratio. We have also evaluated the use of internal standards to provide independent confirmation of the variance in fold-changes at the peptide level, and therefore the variance in protein quantification. Ion Mobility Mass Spectrometry for Detection of Ion Complexes. The Facility accepted delivery and installation of the first commercial Ion Mobility Q-TOF LC/MS instrument (Agilent Technologies, Model 6560) earlier this year. The goal of implementing ion mobility spectrometry (IMS) prior to mass analysis is to add a dimension of separation to sample analysis that is orthogonal to both chromatography and mass spectrometry. Since the IMS operates on a millisecond time scale, compared to the time scale of minutes for chromatography, the device offers the possibility of performing separations of complex mixtures at a much higher rate than possible otherwise. In addition, since IMS separations are associated with collision cross section (CCS) of ions (CCS is essentially a 'shape'parameter of ions in the gas phase), molecules of identical molecular weights can potentially be separated from one another on the basis of their CCS. This has implications for separations of isobaric steroids, lipids, peptides and proteins. IMS also offers the possibility of studying intermolecular complexes and their stoichiometry. One of the initial studies being undertaken is to investigate cyclodextrin-cholesterol complexes, and preliminary results of these measurements suggest a trimeric complex that incorporates calcium ions acting as a bridge. Characterization of Protein Post-translational Modifications. We have characterized post-translational modification of Lys51 in recombinant eIF5A by measuring the intact protein molecular weight by LC/Q-TOF mass spectrometry. This specific Lys is modified in a two-step enzymatic process to form hypusine. Deconvolution of the multiple charge states of the intact protein could detect both modification of Lys51 to deoxy-hypusine and hypusine. The effect of site-specific mutations in eIF5A on altering the level of Lys51 modification were also examined. In a separate project, N-linked glycosylation of recombinant human tyrosinase was characterized. For this recombinant protein, five of the potential seven Asn glycosylation sites were observed. From these sites, eight specific glycopeptides were observed and mapped to three specific Asn residues. Identification of LECT2-associated Amyloidosis in Adrenal Tissue. We recently identified leukocyte cell-derived chemotaxin-2(LECT2) as a component in the formation of amyloid plaques in adrenal tissue. Although adrenal tissue was positive for amyloid by Congo Red staining, specific immunostaining for proteins commonly known to form amyloid plaques were all negative. Plaque proteins from disease tissue were extracted and separated by1D SDS/PAGE. After digestion of the gel bands, serum amyloid P-component and leukocyte cell-derived chemotaxin-2 (LECT2) were identified as components of these plaques. LECT2 has been reported to be found in amyloid plaques in kidney, but this is the first observation of this protein causing amyloidosis in adrenal tissue. The high accumulation of LECT2 in adrenal amyloid plaques from this patient was confirmed by Western blots using a specific LECT2 antibody, which were positive for the disease tissue, while negative for comparable amounts of tissue from normal adrenal glands. Protein Profiling and Quantification in Cerebral Spinal Fluid (CSF) for Disease Biomarkers. We have applied a mass tagging approach to quantitate relative protein expression in CSF from NPC patients, using an 8-plex iTRAQ labeling process to mass tag peptides generated from proteins present in each sample. The method is designed to perform relative protein quantification while maintaining individual patient information and providing the ability to compare results across multiple iTRAQ experiments. Initial studies using the Npc1 knock-out mouse model have been completed to determine the analytical variation of this method. Several differentially expressed proteins identified in this pilot study were also identified in our previous study of differentially expressed proteins in the cerebella of via 2D-GE. This method is now being applied to CSF from patients with the fatal, neurodegenerative, Niemann-Pick Disease, type C1 in order to profile protein changes and identify biochemical alterations correlated to disease progression and treatments. Mass Spectrometric-Based Profiling of Urinary Steroids. Current approaches to the analysis of urinary steroids typically employ either immunoassay or mass spectrometry based technologies. Immunoassay-based methods often lack specificity due to cross-reactivity with other steroids, while targeted LC-MS/MS is limited to the analysis of pre-determined analytes. We have developed a new LC-MS/MS approach to urinary steroid profiling that enables us to detect the steroids that have truly changed in a patient cohort without knowing their identity beforehand (i.e., untargeted metabolomics of steroids). In addition, we have developed a product ion spectrum database of known steroids to improve our capability to identify novel steroids. These methods have been applied to a pilot project to investigate urinary steroids for patients diagnosed with polycystic ovarian syndrome (PCOS). Initially these studies detected elevated levels of an unknown compound consistent with an androgenic steroid in PCOS patients. We were then able to identify the unknown as a mixture of androsterone-sulfate and etiocholanolone-sulfate. We have also developed a reversed phase LC-MRM method to quantitate alpha and beta p-Diol levels in urine. These compounds are intermediates in the backdoor pathway for androgen synthesis. This assay is being applied to studies of patients with Congenital Adrenal Hyperplasia (CAH), and both alpha and beta p-Diol levels in urine were found to correlate with serum androgen levels. This assay is also being applied to studies on PCOS patients.
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