The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface of Arabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho-and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/ dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples.The reversible phosphorylation of specific proteins participates in the regulation of virtually all aspects of cell physiology and development. The extent of its importance is illustrated by the hundreds of conventional protein kinases and phosphatases detected in various eukaryotic genomes (1-3). Whereas, serine, threonine, and tyrosine residues are the typical targets of these kinases, phosphorylation of at least six other amino acids is feasible, potentially expanding even further the dimensions of this post-translational modification (reviewed in Ref. 4). Despite the importance of this pool of phosphorylated proteins, our understanding of its depth and breadth remains sketchy. One barrier has been the lack of methods to define en masse the "phosphorylome," i.e. the subset of proteins in the proteome that become modified in vivo by phosphorylation. Precise characterization of the phosphorylome will be essential to fully understand how proteins are activated or inhibited, encouraged to interact with other components in the cell, and selected for rapid degradation. Certainly, the dynamic and transient nature of many protein phosphorylation reactions underscores the difficulties of resolving the complete phosphorylome for a given organism. Nevertheless, the identification of even just the principal cellular phosphoproteins under dis...

Free-living miracidia of Schistosoma mansoni, upon penetration of the their snail intermediate host, undergo dramatic morphological and physiological changes as they transform to the parasitic sporocyst stage. During this transformation process, developing larvae release a diverse array of proteins, herein referred to as larval transformation proteins (LTPs), some of which are postulated to serve a parasite protective function. In the present study, nanoLC-tandem MS analysis was performed on all proteins represented in entire 1-dimensional SDS-PAGE-separated samples in order to gain a more comprehensive picture of the protein constituents associated with miracidium-tosporocyst transformation and thus, their potential role in influencing establishment of intramolluscan infections. Of 127 proteins with sufficient peptide/sequence information, specific identifications were made for 99, while 28 represented unknown or hypothetical proteins. Nineteen percent of identified proteins possessed signal peptides constituting a cohort of classical secretory proteins, while 22% were identified as putative nonclassically-secreted leaderless proteins based on SecretomeP analysis. Proteins comprising these groups consisted mainly of proteases/protease inhibitors, small HSPs, redox/antioxidant enzymes, ion-binding proteins including those with antioxidant Fe-binding activities (ferritins, heme-binding protein), and venom allergen-like (VAL) proteins. A polyclonal antibody generated against whole LTPs recognized proteins primarily associated with the cilia, ciliated epidermal plates and intercellular ridges of miracidia and the tegument of fully-transformed sporocysts, identifying these structures as sources of a subset of LTPs. Thus lysis of plates and/or leakage during formation of the sporocyst syncytium likely represent significant contributors to the overall LTP makeup, especially identified nonsecretory proteins. However, as plate release/degradation and tegument formation are part of the normal developmental process, all LTPs regardless of tissue origin, would be expected at the parasite-host interface upon infection. This study significantly expands the repertoire of LTPs associated with larval transformation and identifies several, e.g., those involved in stress responses, proteolysis/inhibition, antioxidant and detoxication, and immune modulation, that may play a parasite protective role during this crucial period of transition.

A major limitation of electrospray ionization mass spectrometry (ESI-MS) for oligonucleotide analysis arises due to sodium adduction, a problem that increases with molecular weight. Sodium adduction can preclude useful measurements when limited sample sizes prevent off-line cleanup. A novel and generally useful on-line microdialysis technique is described for the rapid (∼1-5 min) DNA sample cleanup for ESI-MS. Mass spectra of oligonucleotides of different size and sequence showing no significant sodium adduct peaks were obtained using the on-line microdialysis system with sodium chloride concentrations as high as 250 mM. Signal-to-noise ratios were also greatly enhanced compared to direct infusion of the original samples. By using ammonium acetate as the dialysis buffer, it was also found that the noncovalent association of double-stranded oligonucleotides could be preserved during the microdialysis process, allowing analysis by ESI-MS.

In recent years a variety of quantitative proteomics techniques have been developed, allowing characterization of changes in protein abundance in a variety of organisms under various biological conditions. Because it allows excellent control for error at all steps in sample preparation and analysis, full metabolic labeling using Overall full metabolic labeling and partial metabolic labeling prove to be comparable with respect to dynamic range, accuracy, and reproducibility, although partial metabolic labeling consistently allows quantification of a higher percentage of peptide observations across the dynamic range. This difference is especially pronounced at extreme ratios. Ultimately both full metabolic labeling and partial metabolic labeling prove to be well suited for quantitative proteomics characterization.

We report the first metabolic labeling of Arabidopsis thaliana for proteomic investigation, demonstrating efficient and complete labeling of intact plants. Using a reversed-database strategy, we evaluate the performance of the MASCOT search engine in the analysis of combined natural abundance and 15N-labeled samples. We find that 15N-metabolic labeling appears to increase the ambiguity associated with peptide identifications due in part to changes in the number of isobaric amino acids when the isotopic label is introduced. This is reflected by changes in the distributions of false positive identifications with respect to MASCOT score. However, by determining the nitrogen count from each pair of labeled and unlabeled peptides we may improve our confidence in both heavy and light identifications.

Assignment of individual compound identities within mixtures of thousands of metabolites in biological extracts is a major challenge for metabolomic technology. Mass spectrometry offers high sensitivity over a large dynamic range of abundances and molecular weights but is limited in its capacity to discriminate isobaric compounds. In this article, we have extended earlier studies using isotopic labeling for elemental composition elucidation (Rodgers, R. P.; Blumer, E. N.; Hendrickson, C. L.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2000, 11, 835-40) to limit the formulas consistent with any exact mass measurement by comparing observations of metabolites extracted from Arabidopsis thaliana plants grown with (I) (12)C and (14)N (natural abundance), (II) (12)C and (15)N, (III) (13)C and (14)N, or (IV) (13)C and (15)N. Unique elemental compositions were determined over a dramatically enhanced mass range by analyzing exact mass measurement data from the four extracts using two methods. In the first, metabolite masses were matched with a library of 11,000 compounds known to be present in living cells by using values calculated for each of the four isotopic conditions. In the second method, metabolite masses were searched against masses calculated for a constrained subset of possible atomic combinations in all four isotopic regimes. In both methods, the lists of elemental compositions from each labeling regime were compared to find common formulas with similar retention properties by HPLC in at least three of the four regimes. These results demonstrate that metabolic labeling can be used to provide additional constraints for higher confidence formula assignments over an extended mass range.

Typical mass spectrometry-based protein lists from purified fractions are confounded by the absence of tools for evaluating contaminants. In this report, we compare the results of a standard survey experiment using an ion trap mass spectrometer with those obtained using dual isotope labeling and a Q-TOF mass spectrometer to quantify the degree of enrichment of proteins in purified subcellular fractions of Arabidopsis plasma membrane. Incorporation of a stable isotope, either H 2

Objective The first aim was to investigate specific signature patterns of metabolites that are significantly altered in first-trimester serum of women who subsequently developed preeclampsia (PE) compared to healthy pregnancies. The second aim of this study was to examine the predictive performance of the selected metabolites for both early onset [EO-PE] and late onset PE [LO-PE].MethodsThis was a case-control study of maternal serum samples collected between 8+0 and 13+6 weeks of gestation from 167 women who subsequently developed EO-PE n = 68; LO-PE n = 99 and 500 controls with uncomplicated pregnancies. Metabolomics profiling analysis was performed using two methods. One has been optimized to target eicosanoids/oxylipins, which are known inflammation markers and the other targets compounds containing a primary or secondary biogenic amine group. Logistic regression analyses were performed to predict the development of PE using metabolites alone and in combination with first trimester mean arterial pressure (MAP) measurements.ResultsTwo metabolites were significantly different between EO-PE and controls (taurine and asparagine) and one in case of LO-PE (glycylglycine). Taurine appeared the most discriminative biomarker and in combination with MAP predicted EO-PE with a detection rate (DR) of 55%, at a false-positive rate (FPR) of 10%.ConclusionOur findings suggest a potential role of taurine in both PE pathophysiology and first trimester screening for EO-PE.