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Fruiting body formation of Myxococcus xanthus, like biofilm formation of many other organisms, involves the production of an extracellular matrix (ECM). While the polysaccharide component has been studied, the protein component has been largely unexplored. Proteins associated with the ECM were solubilized from purified ECM by boiling with sodium dodecyl sulfate and were identified by liquid chromatography-tandem mass spectrometry of tryptic fragments. The ECM is enriched in proteins of novel function; putative functions were assigned for only 5 of the 21 proteins. Thirteen putative ECM proteins had lipoprotein secretion signals. The genes for many ECM proteins were disrupted in the wild-type (WT), fibA, and pilA backgrounds. Disruption of the MXAN4860 gene had no effect in the WT or fibA baekground but in the pilA background resulted in a 24-h delay in aggregation and sporulation compared to its parent. The results of this study show that the M. xanthus ECM proteome is diverse and novel.

Deuterium for hydrogen exchange at amide sites in proteins is a well-established means of probing the stability of certain proteins and the effects of interactions with ligands and other proteins. When deuterium content is analyzed by mass spectrometry (MS) of digested peptides, corrections frequently need to be made for back-exchange that occurs during digestion, separation, and analysis. The back-exchange process is actually complex and deserving of analysis in a sequence-specific manner. Here an analysis of back-exchange in the decapeptide, angiotensin I, and a hexapeptide derived by digestion of a [sup.15]N-labeled carbohydrate-binding protein, galectin-3, is presented. Nuclear magnetic resonance (NMR) data are used to study back-exchange at specific sites in typical solvents used for separation and analysis, and the derived rates are found to be predictable using methods established for aqueous solvents. The predictability provides potentially new means of improving site specificity of MS analysis and new means of assigning NMR resonances for deuterium content analysis in peptides.

Research has been conducted on Aspergillus niger enzyme endopolygalacturonase II. The use of amide exchange mass spectrometry in investigating this enzyme as it binds to the oligosaccharide substrate and the endopolygalacturonase II inhibition mechanism are described.

Research has been conducted on the inositol triphosphate-(InsP (sub)3 -) gated Ca channels. The association of the cerebellum InsP (sub)3 receptor with InsP (sub)3 binding inhibitor has been studied.

The surface of a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) target can be covalently modified so that it behaves like a medium for solid-phase extraction (SPE). These modified targets are capable of binding molecules of interest, but not contaminants, from sample solutions placed on them. This allows the analyte to be cleaned up on the probe surface by simply washing the target to remove the contaminants prior to MALDI-MS analysis. A limitation of the current SPE/MALDI-MS targets is that they have a fairly low binding capacity, since the coating on these targets is based upon serf-assembled monolayers (SAMs). To overcome this limitation, we have investigated new surface coatings for SPE/MALDI-MS that will have a higher binding capacity than targets modified with SAMs. Here, we describe the development of new SPE/MALDI-MS surfaces that have very high molecular weight (>300 000) polylysine chains attached to them. Targets modified in this manner are capable of binding peptides/proteins by ion-pairing interactions and have approximately 100 times the binding capacity of the SAM-based targets. Furthermore, these polylysine targets can capture over 60% of a protein from a highly contaminated solution. Consequently, polylysine SPE/MALDI-MS targets offer a practical solution for analyzing very small volumes (

One method used to determine the primary sequence of oligosaccharides is to digest them with exoglycosidases and analyze the resulting digestion products by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Previous research has demonstrated that these digestions can be performed on the MALDI target. However, the procedure requires the sample to be incubated at elevated temperatures, and complete digestion requires a few hours. We demonstrate new conditions that permit exoglycosidase digestions to be performed on the MALDI target at room temperature within 30 min. Oligosaccharide standards were digested with one or more exoglycosidases to show that the enzymes retain their activity and specificity under these new reaction conditions. Using this method, the primary sequences of carbohydrate chains can be determined in a relatively short amount of time.

We have developed a new strategy to perform matrix-assisted laser desorption/ionization (MALDI) on magnetic sector instruments equipped with nonintegrating point detectors. This approach is based on using liquid matrices to provide very long-lasting analyte ion currents and to overcome the inability of these instruments to simultaneously detect ions over a range of mass-to-charge values. Here, we demonstrate some of the capabilities of MALDI on a magnetic sector for analyzing biological samples. These capabilities include the ability to perform MS/MS experiments, to attain resolutions over 8000 (full width at half-maximum), and to determine relative molecular masses with < 20 ppm error for a single measurement and

The use of continuousflow fast atom bombardment (CF-FAB) to ionize the sample for tandem mass spectrometry (MS/MS) reduces the background ion current in the sequencing of peptides at the picomole level, thus improving the signal/noise ratio 50 to 100times. The required amount of analyte drops by 25-75%. When paired with array detection (AD), the method registers a detection limit of 900 fmol, well below that of the sequential Edman method. Moreover, CF-FAB-AD-MS/MS can analyze peptide samples at levels where conventional FAB methods cannot yield detectable ions.

Abstract Background Chronic chagasic cardiomyopathy is a debilitating and frequently fatal outcome of human infection with the protozoan parasite, Trypanosoma cruzi. Microarray analysis of gene expression during the T. cruzi life-cycle could be a valuable means of identifying drug and vaccine targets based on their appropriate expression patterns, but results from previous microarray studies in T. cruzi and related kinetoplastid parasites have suggested that the transcript abundances of most genes in these organisms do not vary significantly between life-cycle stages. Results In this study, we used whole genome, oligonucleotide microarrays to globally determine the extent to which T. cruzi regulates mRNA relative abundances over the course of its complete life-cycle. In contrast to previous microarray studies in kinetoplastids, we observed that relative transcript abundances for over 50% of the genes detected on the T. cruzi microarrays were significantly regulated during the T. cruzi life-cycle. The significant regulation of 25 of these genes was confirmed by quantitative reverse-transcriptase PCR (qRT-PCR). The T. cruzi transcriptome also mirrored published protein expression data for several functional groups. Among the differentially regulated genes were members of paralog clusters, nearly 10% of which showed divergent expression patterns between cluster members. Conclusion Taken together, these data support the conclusion that transcript abundance is an important level of gene expression regulation in T. cruzi. Thus, microarray analysis is a valuable screening tool for identifying stage-regulated T. cruzi genes and metabolic pathways.