Biomedical subjects

Kudzai E Mutenda

Publications and source records attributed to Kudzai E Mutenda.

7 recordsLinked to original sources

PubMed2007

Differential fragmentation patterns of pectin oligogalacturonides observed by nanoelectrospray quadrupole ion-trap mass spectrometry using automated spectra interpretation.

Oligogalacturonides of different degrees of polymerization (DP) and methyl esterification (DE) were structurally analyzed by nanoESI quadrupole ion-trap mass spectrometry. The fragmentation patterns of the oligogalacturonides were compared using the program 'Virtual Expert Mass Spectrometrist' (VEMS) for structural annotation. In the analyzed oligogalacturonides of lower DP, the generation of C/Y ions, i.e. ions retaining the glycosidic oxygen, was higher than that of B/Z ions. In general, with oligogalacturonides of higher DP, the B/Z ions were generated more abundantly. Oligogalacturonides with free carboxylic acid groups underwent higher water loss compared to fully methyl-esterified oligogalacturonides under the same fragmentation conditions. Cross-ring cleavage, in which fragmentation occurs across the ring system of the galacturonate residue and signified by unique mass losses, was observed to be higher in fully methyl-esterified oligogalacturonides than in non-methyl-esterified ones. This study demonstrates the different fragmentation patterns of oligogalacturonides as influenced by the presence or absence of methyl ester groups. For a detailed analysis of unknown oligogalacturonides, cross-ring fragmentation gives more structural information than glycosidic bond cleavage. One implication of this is that more structural information is obtained when analyzing methyl-esterified oligogalacturonides than non-methyl-esterified ones in an ion-trap instrument. This is of particular importance in pectin chemistry, where mass spectrometry has become the technique of choice for structural determination. Although this study was not designed to explain the mechanisms of oligogalacturonide fragmentation, possible explanations for why non-methyl-esterified oligogalacturonides undergo more water loss than methyl-esterified ones will be postulated. In addition, the VEMS program was extended to automatically interpret and assign the fragment ions peaks generated in this study.

Nanotechnology↗

PubMed2007

Introduction to proteomics.

Mass spectrometry (MS) has recently become one of the most informative methods for studying proteins. Albiet, MS cannot compete with the detailed structural information obtained by methods such as nuclear magnetic resonance and X-ray crystallography. However, MS is much easier to automate and use as a large-scale technique. Large-scale proteomic methods are valuable for studying the dynamics of the proteins and their posttranslational modification in living cells. Despite the great potential of mass spectrometers, many laboratories are struggling with data analysis and data storage. The complexity of the data analysis stems from the large number of experiments that can be performed by various mass spectrometers. In addition, many mass spectrometers have their own data formats. Performing data analysis on MS data, therefore, requires a rather extensive setup of algorithms and data parsers. In recent years it has become evident that the proteomics society needs standard formats for storing and exchanging data. This has triggered a new problem, which is the invention of several different standard formats. In this chapter, an overview of the most common proteomics experiments with MS, together with an overview of data formats, is presented.

Amino Acid Sequence↗

PubMed2007

Analysis of carbohydrates by mass spectrometry.

The analysis method described in this chapter demonstrates the structural characterization of carbohydrates based on their molecular mass, as well as the mass of their respective fragment ions using mass spectrometry (MS). The carbohydrate molecules are first converted into gaseous ions, under vacuum, after which their mass-to-charge ratio is measured. The mass-to-charge ratio provides information on their preliminary identification, which is further elucidated by fragmenting the ions under a process of collision-induced dissociation. The masses obtained in the first stage of MS together with those obtained in the subsequent stages (MSn) are combined into a mass list that is loaded into the program, Virtual Expert Mass Spectrometrist (VEMS) v3.0. The mass lists obtained are then used by VEMS to search a database of glycans to give the identity of the carbohydrate and the correct assignment of the fragment ions.

Carbohydrates↗

PubMed2005

Fractionation, solid-phase immobilization and chemical degradation of long pectin oligogalacturonides. Initial steps towards sequencing of oligosaccharides.

This work presents the optimized separation of pectin oligomers, their analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), their subsequent immobilization to supports, and our initial steps towards solid-support assisted sequencing. The ambient pressure strong anion-exchange resin Source 15Q combined with ammonium formate buffer (AF) was used for the separation of unsaturated and saturated pectic oligogalacturonides (OGAs) derived from enzymatic digestion of pectin. Routinely, multi-milligram quantities of defined sizes OGAs with DPs from 5 to 19 were produced in excellent purity (>95%). Elution of OGAs followed by direct analysis of the peak fractions by MALDI-TOF MS. Purified OGAs (DP 5-7) were chemoselectively immobilized onto aminooxy-terminated polyethylene glycol polyacrylamide (PEGA) supports. Solid-phase anchoring took place at the reducing end of the oligosaccharide and resulted in the formation of an oxime linkage. The very high coupling yields confirmed the general suitability of aminooxy-PEGA resins for the immobilization of OGAs of different lengths. The OGA-functionalized PEGA supports were subsequently treated with aq TFA at 40 or 60 degrees C, and the chemical degradation products released from the support were analyzed by ESIMS. In all cases, the original OGA was degraded into smaller oligomers of various sizes down to the monomer. This work illustrates some of the basic principles underlying a strategy ultimately aimed at solid-support assisted sequencing of oligosaccharides.

Carbohydrate Sequence↗

PubMed2005

Alpha-glucan, water dikinase (GWD): a plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity.

The recently discovered potato tuber (Solanum tuberosum) alpha-glucan, water dikinase (GWD) (formerly known as R1) catalyzes the phosphorylation of starch by a dikinase-type reaction mechanism in which the beta-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glucosyl residue of starch. In the present study, we found that the GWD enzyme is inactive in the oxidized form, which is accompanied by the formation of a specific intramolecular disulfide bond as determined by disulfide-linked peptide mapping. The regulatory properties of this disulfide linkage were confirmed by site-directed mutagenesis studies. Both reduced thioredoxin (Trx) f and Trx m from spinach leaves reduced and activated oxidized GWD at very low concentrations, with Trx f being the more efficient, yielding an S0.5 value of 0.4 microM. Interestingly, GWD displays a reversible and selective binding to starch granules depending on the illumination state of the plant. Here we show that starch granule-bound GWD isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced Trx. Furthermore, the soluble form of GWD was found in its fully reduced state, providing evidence of a Trx-controlled regulation mechanism linking enzymatic activity and specific binding affinities of a protein to an intracellular surface. The regulatory site sequence, CFATC, of potato GWD is conserved in chloroplast-targeted GWDs from other species, suggesting an overall redox regulation of the GWD enzyme.

Amino Acid Sequence↗

PubMed2005

Identification of novel lysosomal matrix proteins by proteome analysis.

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.

Animals↗

PubMed2002

Application of mass spectrometry to determine the activity and specificity of pectin lyase A.

Electrospray ionization (ESI) with quadrupole ion-trap mass spectrometry was used to assess the activity and specificity of the enzyme pectin lyase A (PLA) (EC 4.2.2.10) on model pectins with varying degrees and patterns of methyl esterification. PLA is a pectinase which cleaves alpha-(1-->4)-glycosidic linkages in pectin by a trans-elimination process. Using pectins with different degrees and patterns of methyl esterification, there was a significant variation in the activity rate of PLA. The enzymatic products generated at various time intervals were structurally analyzed by mass spectrometry to determine the specificity of PLA. Although the preferred substrate for PLA is fully methyl esterified polygalacturonate, cleavage was also observed with a non-methyl esterified galacturonic acid residue on either the non-reducing end or the reducing end. The current study shows that although PLA prefers fully methyl esterified substrates it can also accept partially esterified ones. It also demonstrates the suitability of ESI ion-trap mass spectrometry in determining enzyme specificities.

Carbohydrate Sequence↗