Strategies for biomarker discovery increasingly focus on biofluid protein and peptide expression patterns. Posttranslational modifications contribute significantly to the pattern complexity and thereby increase the likelihood of obtaining specific biomarkers for diagnostics and disease monitoring. Glycosylation is a common post-translational modification that plays a role e.g. in cell adhesion and in cell-cell and receptor-ligand interactions. Abnormal protein glycosylation has important disease associations, and the glycoproteome is therefore a target for biomarker discovery. Here we present a simple and highly selective strategy for purification of sialic acid-containing glycopeptides (the sialiome) from complex peptide mixtures. The approach utilizes a high and selective affinity of sialic acids for titanium dioxide under specific buffer conditions. In combination with mass spectrometry we used this strategy to characterize the human plasma and saliva sialiomes where 192 and 97 glycosylation sites, respectively, were identified. Furthermore we illustrate the potential of this method in biomarker discovery.

Centrosomes in animal cells are dynamic organelles with a proteinaceous matrix of pericentriolar material assembled around a pair of centrioles. They organize the microtubule cytoskeleton and the mitotic spindle apparatus. Mature centrioles are essential for biogenesis of primary cilia that mediate key signalling events. Despite recent advances, the molecular basis for the plethora of processes coordinated by centrosomes is not fully understood. We have combined protein identification and localization, using PCP-SILAC mass spectrometry, BAC transgeneOmics, and antibodies to define the constituents of human centrosomes. From a background of non-specific proteins, we distinguished 126 known and 40 candidate centrosomal proteins, of which 22 were confirmed as novel components. An antibody screen covering 4000 genes revealed an additional 113 candidates. We illustrate the power of our methods by identifying a novel set of five proteins preferentially associated with mother or daughter centrioles, comprising genes implicated in cell polarity. Pulsed labelling demonstrates a remarkable variation in the stability of centrosomal protein complexes. These spatiotemporal proteomics data provide leads to the further functional characterization of centrosomal proteins.

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.

Sixteen (1.5%) of the 1,043 clinical macrolide-resistant Streptococcus pneumoniae isolates collected and analyzed in the 1999-2000 PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study have resistance mechanisms other than rRNA methylation or efflux. We have determined the macrolide resistance mechanisms in all 16 isolates by sequencing the L4 and L22 riboprotein genes, plus relevant segments of the four genes for 23S rRNA, and the expression of mutant rRNAs was analyzed by primer extension. Isolates from Canada (n ‫؍‬ 4), Japan (n ‫؍‬ 3), and Australia (n ‫؍‬ 1) were found to have an A2059G mutation in all four 23S rRNA alleles. The Japanese isolates additionally had a G95D mutation in riboprotein L22; all of these originated from the same collection center and were clonal. Three of the Canadian isolates were also clonal; the rest were not genetically related. Four German isolates had A2059G in one, two, and three 23S rRNA alleles and A2058G in two 23S rRNA alleles, respectively. An isolate from the United States had C2611G in three 23S rRNA alleles, one isolate from Poland had A2058G in three 23S rRNA alleles, one isolate from Turkey had A2058G in four 23S rRNA alleles, and one isolate from Canada had A2059G in two 23S rRNA alleles. Erythromycin and clindamycin resistance gradually increased with the number of A2059G alleles, whereas going from one to two mutant alleles caused sharp rises in the azithromycin, roxithromycin, and rokitamycin MICs. Comparisons of mutation dosage with rRNA expression indicates that not all alleles are equally expressed. Despite their high levels of macrolide resistance, all 16 isolates remained susceptible to the ketolide telithromycin (MICs, 0.015 to 0.25 g/ml).Macrolide, lincosamide, and streptogramin B (MLS B ) resistance in Streptococcus pneumoniae occurs either by modification of the drug-binding site or by active efflux of the drug. Target modification is usually the result of dimethylation of the adenine residue at position 2058 on the 23S rRNA by a methylase enzyme (30). In S. pneumoniae, Erm(B) [encoded by the erm(B) gene] is the enzyme mostly responsible (7, 30), although, more rarely, a methylase encoded by the erm(A) subclass erm(TR) gene is implicated (7,23).In vitro studies have demonstrated that target modification can also be achieved via mutations in domains II and V of 23S rRNA and in the genes encoding riboproteins L4 and L22 and can confer macrolide, lincosamide, streptogramin, and ketolide resistance (2, 24). Although previous reports are rare, such mutations have been found in MLS B -resistant clinical isolates (3,14,25). However, until now there have been no studies on the prevalence and epidemiology of these types of mutations in clinical isolates on a worldwide scale.PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) is a longitudinal, global, multicenter surveillance study of respiratory tract pathogens. We screened all macrolide-resistant S. pneumoniae isolat...