This study encompasses a collection of experiences with regard to numerous matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation techniques in terms of their suitability for di †erent peptide and protein analytes. Variants of both established and new sample preparation techniques for the MALDI-MS analysis of peptides and proteins are described. The importance of matrix selection, matrix and analyte concentration, pH adjustment, crystallization conditions and the use of additives is evaluated. The tolerance of the di †erent sample preparations towards salts, bu †ers, synthetic polymers, detergents, denaturants and other contaminants, and also the inÑuence of the preparation methods on undesired amino acid side-chain oxidation, are investigated. Moreover, the performance of on-target tryptic digestion and on-target disulÐde reduction is shown and a microscale puriÐcation procedure is described.

This study encompasses a collection of experiences with regard to numerous matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) sample preparation techniques in terms of their suitability for different peptide and protein analytes. Variants of both established and new sample preparation techniques for the MALDI‐MS analysis of peptides and proteins are described. The importance of matrix selection, matrix and analyte concentration, pH adjustment, crystallization conditions and the use of additives is evaluated. The tolerance of the different sample preparations towards salts, buffers, synthetic polymers, detergents, denaturants and other contaminants, and also the influence of the preparation methods on undesired amino acid side‐chain oxidation, are investigated. Moreover, the performance of on‐target tryptic digestion and on‐target disulfide reduction is shown and a microscale purification procedure is described. According to this study, there is no universally applicable sample preparation for a broad variety of analytes. Rather, it is necessary to specifically adapt the sample preparation to the analyte properties. © 1997 John Wiley & Sons, Ltd.

Glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) are required for normal starch metabolism. We analysed starch phosphorylation in Arabidopsis wildtype plants and mutants lacking either GWD or PWD using 31 P NMR. Phosphorylation at both C6-and C3-positions of glucose moieties in starch was drastically decreased in GWD-deficient mutants. In starch from PWD-deficient plants C3-bound phosphate was reduced to levels close to the detection limit. The latter result contrasts with previous reports according to which GWD phosphorylates both C6-and C3-positions. In these studies, phosphorylation had been analysed by HPLC of acidhydrolysed glucans. We now show that maltose-6-phosphate, a product of incomplete starch hydrolysis, co-eluted with glucose-3-phosphate under the chromatographic conditions applied. Re-examination of the specificity of the dikinases using an improved method demonstrates that C6-and C3-phosphorylation is selectively catalysed by GWD and PWD, respectively.

Eukaryotic cells are composed of a variety of membrane-bound organelles that are thought to derive from symbiotic associations involving bacteria, archaea, or other eukaryotes. In addition to acquiring the plastid, all Archaeplastida and some of their endosymbiotic derivatives can be distinguished from other organisms by the fact that they accumulate starch, a semicrystalline-storage polysaccharide distantly related to glycogen and never found elsewhere. We now provide the first evidence for the existence of starch in a particular species of single-cell diazotrophic cyanobacterium. We provide evidence for the existence in the eukaryotic host cell at the time of primary endosymbiosis of an uridine diphosphoglucose (UDP-glucose)-based pathway similar to that characterized in amoebas. Because of the monophyletic origin of plants, we can define the genetic makeup of the Archaeplastida ancestor with respect to storage polysaccharide metabolism. The most likely enzyme-partitioning scenario between the plastid's ancestor and its eukaryotic host immediately suggests the precise nature of the ancient metabolic symbiotic relationship. The latter consisted in the export of adenosine diphosphoglucose (ADP-glucose) from the cyanobiont in exchange for the import of reduced nitrogen from the host. We further speculate that the monophyletic origin of plastids may lie in an organism with close relatedness to present-day group V cyanobacteria.

SummaryAmong the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.