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The quantification of phosphate bound to the C6 and C3 positions of glucose residues in starch has received increasing interest since the importance of starch phosphorylation for plant metabolism was discovered. The method described here is based on the observation that the isobaric compounds glucose-6-phosphate (Glc6P) and glucose-3- phosphate (Glc3P) exhibit significantly different fragmentation patterns in negative ion electrospray tandem mass spectrometry (MS/MS). A simple experiment involving collision-induced dissociation (CID) MS2 spectra of the sample and the two reference substances Glc3P and Glc6P permitted the quantification of the relative amounts of the two compounds in monosaccharide mixtures generated by acid hydrolysis of starch. The method was tested on well-characterized potato tuber starch. The results are consistent with those obtained by NMR analysis. In contrast to NMR, however, the presented method is fast and can be performed on less than 1 mg of starch. Starch samples of other origins exhibiting a variety of phosphorylation degrees were analyzed to assess the sensitivity and robustness of the method.
Heterochitooligosaccharides possess interesting biol. properties. Isobaric mixts. of such linear heterochitooligosaccharides can be obtained by chem. or enzymic degrdn. of chitosan. However, the sepn. of such mixts. is a challenging anal. problem which is so far unresolved. It is shown that these isobaric mixts. can be sequenced and quantified simultaneously using std. derivatization and multistage tandem mass spectrometric techniques. A linear ion trap mass spectrometer equipped with a vacuum matrix-assisted laser desorption ionization (vMALDI) source is used to perform MS2 as well as MS3 expts. [on SciFinder (R)].
Background: Transthyretin (TTR), a traditional biomarker for nutritional and inflammatory status exists in different molecular variants of yet unknown importance. A truncated form of TTR has recently been described to be part of a set of biomarkers for the diagnosis of ovarian cancer. The main aim of the study was therefore to characterize differences in microheterogeneity between ascitic fluid and plasma of women affected with ovarian cancer and to evaluate the tumor site as the possible source of TTR. Methods: Subjects were 48 women with primary invasive epithelial ovarian cancer or recurrent ovarian carcinoma. The control group consisted of 20 postmenopausal women. TTR and retinol-binding protein (RBP) levels were measured by enzyme-linked immunoassay ( ELISA) and C-reactive protein (CRP) levels by a high- sensitivity latex particle turbidimetric assay. The molecular heterogeneity of TTR was analysed using immunoprecipitation and matrix-associated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Presence of TTR in tumor tissue was determined with indirect peroxidase immunostaining. Results: TTR and RBP (mu g/ml) levels in serum were 148.5 +/- 96.7 and 22.5 +/- 14.8 in affected women compared to 363.3 +/- 105.5 and 55.8 +/- 9.3 in healthy postmenopausal women ( p < 0.01). In ascitic fluid, levels were 1.02 +/- 0.24 and 4.63 +/- 1.57 mu g/ml, respectively. The mean levels of TTR and RBP in serum showed a tendency to decrease with the severity of the disease and were lower in affected women whose CRP levels were > 40 mg/ml ( p = 0.08 for TTR; p < 0.05 for RBP). No differences in TTR microheterogeneity were observed between TTR isolated from serum of affected and healthy women or from ascitic fluid. TTR occurred rather consistently in four variants. Mass signals were at 13758 +/- 7, 13876 +/- 13 ( greatest intensity), 13924 +/- 21 and 14062 +/- 24 Da, representing native, S-cysteinylated, S-cysteinglycinylated and glutathionylated TTR, respectively. Serum of healthy and affected women as well as ascitic fluid contained the truncated fragment of TTR ( 12828 +/- 11 Da). No immunoreactive TTR was observed in the tumor sites. Conclusion: The severity of the cancer associated catabolism as well as the inflammation status affect serum TTR and RBP levels. Neither TTR nor its truncated form originates from tumor tissue and its occurrence in ascites may well reflect the filtration from blood into ascitic fluid
Skeletal muscle differentiation is a complex process: It is characterised by changes in gene expression and protein composition. Simultaneously, a dramatic remodelling of the cytoskeleton and associated cell-matrix contacts, the costameres, occurs. The expression and localisation of the protein ponsin at cell-matrix contacts marks the establishment of costameres. In this report we show that skeletal muscle cells are characterised by a novel ponsin isoform, which contains a large insertion in its carboxy-terminus. This skeletal muscle-specific module binds the adapter proteins Nck1 and Nck2, and increased co-localisation of ponsin with Nck2 is observed at remodelling cell-matrix contacts of differentiating skeletal muscle cells. Since this ponsin insertion can be phosphorylated, it may adjust the interaction affinity with Nck adapter proteins. The novel ponsin isoform and its interaction with Nck1/2 provide exciting insight into the convergence of signalling pathways at the costameres, and its crucial role for skeletal muscle differentiation and re-generation.