Combining metal oxide affinity chromatography (MOAC) and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites

BACKGROUND: Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. RESULTS: Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC) and selective ion trap mass spectrometry. The complete approach involves (i) enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii) gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment), (iii) identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv) identification of phosphorylation sites of these enriched proteins using automatic detection of H(3)PO(4 )neutral loss peaks and data-dependent MS(3)-fragmentation of the corresponding MS(2)-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. CONCLUSION: A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to proteins extracted from C. reinhardtii. Thus, the method can easily be adapted to suit the sample of interest since it is inexpensive and the components needed are widely available. Reproducibility of the approach was tested by monitoring phosphorylation sites on specific proteins from seeds and C. reinhardtii in duplicate experiments. The whole process is proposed as a strategy adaptable to other plant tissues providing high confidence in the identification of phosphoproteins and their corresponding phosphorylation sites

Comparative proteomics reveal characteristics of life-history transitions in a social insect

<p>Abstract</p> <p>Background</p> <p>Honey bee (<it>Apis mellifera</it>) workers are characterized by complex social behavior. Their life-history is dominated by a period of within-nest activity followed by a phase of long-distance flights and foraging. General insights into insect metabolism imply that foraging onset is associated with fundamental metabolic changes, and theory on social evolution suggests metabolic adaptations that are advantageous for the colony as a whole.</p> <p>Results</p> <p>Here we address the life-history characteristics of workers with LC-MS/MS based relative quantification of major proteins. Our approach includes: i. Calculation of a false positive rate for the identifications, ii. Support of relative protein quantification results obtained from spectral count by non-parametric statistics, and iii. Correction for Type 1 error inflation using a bootstrap iteration analysis. Our data are consistent with the use of glucose as the main fuel for honey bee flight. Moreover, the data delivers information on the expression of ATPsynthases/ATPases, and provide new insights into nurse- and forager-specific patterns of protection against oxidative stress.</p> <p>Conclusion</p> <p>The results show the suitability of this approach to investigate fundamental biochemical changes in an insect, and provide new evidence for metabolic specializations that occur during the social ontogeny of worker honey bees.</p

Characterisation of protein phosphorylation in Arabidopsis thaliana and Chlamydomonas reinhardtii based on affinity chromatography and mass spectrometry

Reversible protein phosphorylation is of key importance for several mechanisms of life. However, the low abundance of phosphorylated proteins hinders investigations following this direction. To circumvent this problem a new method for the enrichment of phosphorylated proteins out of complex protein mixtures was developed. This method relies on the affinity of the phosphate group towards aluminum hydroxide and was termed MOAC (Metal Oxide Affinity Chromatography). Successful phosphoprotein enrichment even from highly complex protein mixtures was confirmed by separation of phosphorylated from non-phosphorylated standard proteins, detection using a phosphate-specific fluorescent stain, ICP-MS (Inductively Coupled Plasma – Mass Spectrometry), and antibodies. To identify phosphopeptides and protein phosphorylation sites proteins were digested with an endoproteinase and subjected to LC/MSn analysis. The determination of protein phosphorylation sites was achieved by making use of the distinct neutral loss of phosphoric acid from peptides phosphorylated on serine or threonine during fragmentation in an ion trap mass spectrometer. However, it was observed that also other posttranslational modifications such as methionine oxidation can mimic phosphorylation when using such data-dependent neutral loss scans. Remedies were defined to circumvent this problem leading to more reliable phosphorylation site identification. The combination of MOAC and a neutral loss driven MS3 approach showed to be highly useful for investigations on plant protein phosphorylation. In one of the first non-targeted phosphoproteomics approaches in plant science this combined approach was applied to enrich phosphoproteins of A. thaliana and of C. reinhardtii leading to the identification of over 40 phosphopeptides, 27 phosphorylation sites, and over 30 phosphoproteins. In addition, over 300 putative phosphoproteins were identified. In a targeted analysis the speculative in vivo phosphorylation site of the metabolic key enzyme phosphoenolpyruvate carboxylase in A. thaliana could be confirmed. While the detection of phosphorylation and the determination of phosphorylation sites are very important to get a first impression whether or not a protein can be influenced by phosphorylation, quantification of phosphorylation delivers more detailed information on protein regulation by phosphorylation. Using an approach involving ICP-MS it was possible to monitor the overall protein phosphorylation degree for different developmental stages of A. thaliana. These values were compared to the degree of phosphorylation of proteins in C. reinhardtii. This comparison shows that protein phosphorylation is most abundant in rapidly dividing tissue and lowest in dormant seeds. To monitor phosphorylation of key metabolic enzymes under different physiological conditions, a robust method for relative quantification of changes in protein phosphorylation on a linear ion trap was also developed. It was used to investigate a speculative connection between temperature and phosphorylation of sucrose-phosphate synthase (SPS) and to determine the stoichiometry of phosphorylation for the in vitro phosphorylated enzyme. The results indicate that temperature has a profound effect on the phosphorylation level of SPS by influencing the activity or abundance of the kinase responsible for SPS phosphorylation

Differential Gene Expression and Protein Abundance Evince Ontogenetic Bias toward Castes in a Primitively Eusocial Wasp

Polistes paper wasps are models for understanding conditions that may have characterized the origin of worker and queen castes and, therefore, the origin of paper wasp sociality. Polistes is “primitively eusocial” by virtue of having context-dependent caste determination and no morphological differences between castes. Even so, Polistes colonies have a temporal pattern in which most female larvae reared by the foundress become workers, and most reared by workers become future-reproductive gynes. This pattern is hypothesized to reflect development onto two pathways, which may utilize mechanisms that regulate diapause in other insects. Using expressed sequence tags (ESTs) for Polistes metricus we selected candidate genes differentially expressed in other insects in three categories: 1) diapause vs. non-diapause phenotypes and/or worker vs. queen differentiation, 2) behavioral subcastes of worker honey bees, and 3) no a priori expectation of a role in worker/gyne development. We also used a non-targeted proteomics screen to test for peptide/protein abundance differences that could reflect larval developmental divergence. We found that foundress-reared larvae (putative worker-destined) and worker-reared larvae (putative gyne-destined) differed in quantitative expression of sixteen genes, twelve of which were associated with caste and/or diapause in other insects, and they also differed in abundance of nine peptides/proteins. Some differentially-expressed genes are involved in diapause regulation in other insects, and other differentially-expressed genes and proteins are involved in the insulin signaling pathway, nutrient metabolism, and caste determination in highly social bees. Differential expression of a gene and a peptide encoding hexameric storage proteins is especially noteworthy. Although not conclusive, our results support hypotheses of 1) larval developmental pathway divergence that can lead to caste bias in adults and 2) nutritional differences as the foundation of the pathway divergence. Finally, the differential expression in Polistes larvae of genes and proteins also differentially expressed during queen vs. worker caste development in honey bees may indicate that regulatory mechanisms of caste outcomes share similarities between primitively eusocial and advanced eusocial Hymenoptera

The Worker Honeybee Fat Body Proteome Is Extensively Remodeled Preceding a Major Life-History Transition

Honeybee workers are essentially sterile female helpers that make up the majority of individuals in a colony. Workers display a marked change in physiology when they transition from in-nest tasks to foraging. Recent technological advances have made it possible to unravel the metabolic modifications associated with this transition. Previous studies have revealed extensive remodeling of brain, thorax, and hypopharyngeal gland biochemistry. However, data on changes in the abdomen is scarce. To narrow this gap we investigated the proteomic composition of abdominal tissue in the days typically preceding the onset of foraging in honeybee workers

Deciphering Proteomic Signatures of Early Diapause in Nasonia

Insect diapause is an alternative life-history strategy used to increase longevity and survival in harsh environmental conditions. Even though some aspects of diapause are well investigated, broader scale studies that elucidate the global metabolic adjustments required for this remarkable trait, are rare. In order to better understand the metabolic changes during early insect diapause, we used a shotgun proteomics approach on early diapausing and non-diapausing larvae of the recently sequenced hymenopteran model organism Nasonia vitripennis. Our results deliver insights into the molecular underpinnings of diapause in Nasonia and corroborate previously reported diapause-associated features for invertebrates, such as a diapause-dependent abundance change for heat shock and storage proteins. Furthermore, we observed a diapause-dependent switch in enzymes involved in glycerol synthesis and a vastly changed capacity for protein synthesis and degradation. The abundance of structural proteins and proteins involved in protein synthesis decreased with increasing diapause duration, while the abundance of proteins likely involved in diapause maintenance (e.g. ferritins) increased. Only few potentially diapause-specific proteins were identified suggesting that diapause in Nasonia relies to a large extent on a modulation of pre-existing pathways. Studying a diapause syndrome on a proteomic level rather than isolated pathways or physiological networks, has proven to be an efficient and successful avenue to understand molecular mechanisms involved in diapause

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Box-and-whisker-plots of proteins homologues to proteins putatively involved in oxygen stress response

<p><b>Copyright information:</b></p><p>Taken from "Comparative proteomics reveal characteristics of life-history transitions in a social insect"</p><p>http://www.proteomesci.com/content/5/1/10</p><p>Proteome Science 2007;5():10-10.</p><p>Published online 17 Jul 2007</p><p>PMCID:PMC1964756.</p><p></p> Boxes represent 25–75% percentiles of the data, outliers are marked as open circles. * denotes significant differences between nest bees and foragers, for p-values see Additional file . CuZn SOD: CuZn superoxide dismutase. GST: glutathione-s-transferase. Spectrum count: spectrum count corrected for actin