A large-scale, targeted metabolomics method for the analysis and quantification of metabolites in human plasma via liquid chromatography-mass spectrometry.

Metabolomics is the study of small molecules, primarily metabolites, that are produced during metabolic processes. Analysis of the composition of an organism's metabolome can yield useful information about an individual's health status at any given time. In recent years, the development of large-scale, targeted metabolomic methods has allowed for the analysis of biological samples using analytical techniques such as LC-MS/MS. This paper presents a large-scale metabolomics method for analysis of biological samples, with a focus on quantification of metabolites found in blood plasma. The method comprises a 10-min chromatographic separation using HILIC and RP stationary phases combined with positive and negative electrospray ionization in order to maximize metabolome coverage. Complete analysis of a single sample can be achieved in as little as 40 min using the two columns and dual modes of ionization. With 540 metabolites and the inclusion of over 200 analytical standards, this method is comprehensive and quantitatively robust when compared to current targeted metabolomics methods. This study uses a large-scale evaluation of metabolite recovery from plasma that enables absolute quantification of metabolites by correcting for analyte loss throughout processes such as extraction, handling, or storage. In addition, the method was applied to plasma collected from adjuvant breast cancer patients to confirm the suitability of the method to clinical samples.

d-Glycero-ß-d-mannoheptose Phosphate 7-O-Modifications.

Pathogen-associated molecular patterns activate the immune system via pattern recognition receptors. Recently, newly discovered pathogen-associated molecular patterns, d-glycero-ß-d-mannoheptose phosphate and d-glycero-ß-d-mannoheptose 1,7-biphosphate, were shown to induce a TRAF-interacting protein with a forkhead-associated domain-dependent immune response in human embryonic kidney cells and colonic epithelial cells. Concurrently, ADP-heptose was shown to bind α-kinase 1 and activate TIFA via phosphorylation leading to an immune cascade to ultimately activate NF-κB. These pathogen-associated molecular patterns have raised interest in the pharmaceutical industry for their potential use as immunomodulators. However, little is understood about the host cell uptake of d-glycero-ß-d-mannoheptose phosphate, d-glycero-ß-d-mannoheptose 1,7-biphosphate, and ADP-heptose in vivo and derivatives of these molecules are needed to interrogate this. In this regard, herein we describe 7-O-modifications of d-glycero-ß-d-mannoheptose phosphate to produce molecular probes toward the development of a useful toolbox for biologists. A convergent strategy that involves introduction of a substituent at O-7 before alkene oxidation was investigated and proved successful in the generation of a range of molecular probes.

Ras transformation results in cleavage of reticulon protein Nogo-B that is associated with impairment of IFN response.

Dysregulation of Ras signaling is the major cause of various cancers. Aberrant Ras signaling, however, provides a favorable environment for many viruses, making them suitable candidates as cancer-killing therapeutic agents. Susceptibility of cancer cells to such viruses is mainly due to impaired type I interferon (IFN) response, often as a result of activated Ras/ERK signaling in these cells. In this study, we searched for cellular factors modulated by Ras signaling and their potential involvement in promoting viral oncolysis. We found that upon Ras transformation of NIH-3T3 cells, the N-terminus of Nogo-B (reticulon 4) was proteolytically cleaved. Interestingly, Nogo knockdown (KD) in non-transformed and Ras-transformed cells both enhanced virus-induced IFN response, suggesting that both cleaved and uncleaved Nogo can suppress IFN response. However, pharmacological blockade of Nogo cleavage in Ras-transformed cells significantly enhanced virus-induced IFN response, suggesting that cleaved Nogo contributes to enhanced IFN suppression in these cells. We further showed that IFN suppression associated with Ras-induced Nogo-B cleavage was distinct from but synergistic with that associated with an activated Ras/ERK pathway. Our study therefore reveals an important and novel role of Nogo-B and its cleavage in the suppression of anti-viral immune responses by oncogenic Ras transformation.

Compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa) rivals that of animals, fungi, and plants.

We present a combined proteomic and bioinformatic investigation of mitochondrial proteins from the amoeboid protist Acanthamoeba castellanii, the first such comprehensive investigation in a free-living member of the supergroup Amoebozoa. This protist was chosen both for its phylogenetic position (as a sister to animals and fungi) and its ecological ubiquity and physiological flexibility. We report 1033 A. castellanii mitochondrial protein sequences, 709 supported by mass spectrometry data (676 nucleus-encoded and 33 mitochondrion-encoded), including two previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Other notable findings include duplicate proteins for all of the enzymes of the tricarboxylic acid (TCA) cycle-which, along with the identification of a mitochondrial malate synthase-isocitrate lyase fusion protein, suggests the interesting possibility that the glyoxylate cycle operates in A. castellanii mitochondria. Additionally, the A. castellanii genome encodes an unusually high number (at least 29) of mitochondrion-targeted pentatricopeptide repeat (PPR) proteins, organellar RNA metabolism factors in other organisms. We discuss several key mitochondrial pathways, including DNA replication, transcription and translation, protein degradation, protein import and Fe-S cluster biosynthesis, highlighting similarities and differences in these pathways in other eukaryotes. In compositional and functional complexity, the mitochondrial proteome of A. castellanii rivals that of multicellular eukaryotes. BIOLOGICAL SIGNIFICANCE: Comprehensive proteomic surveys of mitochondria have been undertaken in a limited number of predominantly multicellular eukaryotes. This phylogenetically narrow perspective constrains and biases our insights into mitochondrial function and evolution, as it neglects protists, which account for most of the evolutionary and functional diversity within eukaryotes. We report here the first comprehensive investigation of the mitochondrial proteome in a member (A. castellanii) of the eukaryotic supergroup Amoebozoa. Through a combination of tandem mass spectrometry (MS/MS) and in silico data mining, we have retrieved 1033 candidate mitochondrial protein sequences, 709 having MS support. These data were used to reconstruct the metabolic pathways and protein complexes of A. castellanii mitochondria, and were integrated with data from other characterized mitochondrial proteomes to augment our understanding of mitochondrial proteome evolution. Our results demonstrate the power of combining direct proteomic and bioinformatic approaches in the discovery of novel mitochondrial proteins, both nucleus-encoded and mitochondrion-encoded, and highlight the compositional complexity of the A. castellanii mitochondrial proteome, which rivals that of animals, fungi and plants.

Data showing the compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa).

This article describes and directly links to 1033 Acanthamoeba castellanii mitochondrial protein sequences. Of these, 709 are supported by Mass Spectrometry (MS) data (676 nucleus-encoded and 33 mitochondrion-encoded). Two of these entries are previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Our analysis corrects many A. castellanii protein sequences that were incorrectly inferred previously from genomic data deposited in NCBI.

Quantitative proteomic investigation employing stable isotope labeling by peptide dimethylation on proteins of strawberry fruit at different ripening stages.

A quantitative proteomic investigation of strawberry fruit ripening employing stable isotope labeling by peptide dimethylation was conducted on 'Mira' and 'Honeoye' strawberry fruit. Postharvest physiological quality indices, including volatile production, total phenolics, total anthocyanins, antioxidant capacity, soluble solids and titratable acidity, were also characterized in white, pink and red fruit. More than 892 and 848 proteins were identified and quantified in the 'Mira' and 'Honeoye' fruit, respectively, using at least two peptides for each protein identification. Using the normalized ratio of protein abundance changes, proteins that changed two-fold or more were identified as proteins that are up- or down-regulated during fruit ripening. Among the quantified proteins, 111 proteins were common to both cultivars and represented five significant clusters based on quantitative changes. Among the up-regulated proteins were proteins involved in metabolic pathways including flavonoid/anthocyanin biosynthesis, volatile biosynthesis, antioxidant metabolism, stress responses and allergen formation. Proteins that decreased during fruit ripening were found to be responsible for methionine metabolism, antioxidant-redox, energy metabolism and protein synthesis. Our results show that strawberry ripening is a highly complex system involving multi-physiological processes made possible through changes in protein expression. This study provides new insights on the regulation of proteins during strawberry fruit ripening that lay the foundation for further targeted studies. BIOLOGICAL SIGNIFICANCE: Research on the postharvest physiology and biochemistry of strawberry fruit as a model of non-climacteric fruit ripening has been conducted for many years. However, the mechanism(s) for the initiation and metabolic regulation of non-climacteric fruit ripening remains unknown. Little information on strawberry fruit ripening is available at the proteome level. This paper is the first report of a quantitative proteomic investigation of strawberry fruit ripening employing stable isotope labeling by peptide dimethylation. Postharvest physiological quality indices, including volatile production, total phenolics, total anthocyanins, antioxidant capacity, soluble solids and titratable acidity, were also characterized in ripening fruit. Significant biological changes associated with ripening were revealed and proteins that change significantly under these conditions were identified. Therefore, our study links the biological events of strawberry fruit ripening with proteomic information and provides insights into possible mechanisms of regulation. Proteins that changed during ripening were analyzed through function analysis, which provides new insights into metabolic changes occurring during ripening. Findings from this paper not only provide proteome information on fruit ripening, but also pave the way for further quantitative studies using SMR to investigate certain proteins and pathways involved in fruit ripening.

A mass spectrometry-based plasma protein panel targeting the tumor microenvironment in patients with breast cancer.

Proteins secreted or shed by cancerous cells are seen as a rich source of biomarkers and novel therapeutic targets. Recently, the importance of the tumor microenvironment, which comprises the surrounding non-tumor cells, has received increased attention for its role in tumor progression. We developed a targeted proteomics assay to monitor a panel of plasma proteins postulated to be present in the tumor microenvironment. The plasma of 76 breast cancer patients was depleted of abundant circulating proteins, enzymatically digested and labeled by reductive methylation. The labeled digests were analyzed by tandem mass spectrometry using a multiple reaction monitoring acquisition method. The protein targets were correlated with the tumor characteristics, the extent of the disease and the clinical staging of the patients. Linear discriminant analysis revealed that infiltrating ductal and invasive mammary breast carcinomas could be grouped based on distinctive peptide levels of fibronectin, clusterin, gelsolin and α-1-microglobulin/Inter-α-trypsin inhibitor light chain precursor (AMBP). These proteins have been previously associated with breast cancer at the tissue level, however, this is the first study to measure plasma levels of these proteins and correlate these levels with clinical features. Significant variability was seen between unique peptides belonging to the same protein. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Composition of the mitochondrial electron transport chain in acanthamoeba castellanii: structural and evolutionary insights.

The mitochondrion, derived in evolution from an α-proteobacterial progenitor, plays a key metabolic role in eukaryotes. Mitochondria house the electron transport chain (ETC) that couples oxidation of organic substrates and electron transfer to proton pumping and synthesis of ATP. The ETC comprises several multiprotein enzyme complexes, all of which have counterparts in bacteria. However, mitochondrial ETC assemblies from animals, plants and fungi are generally more complex than their bacterial counterparts, with a number of 'supernumerary' subunits appearing early in eukaryotic evolution. Little is known, however, about the ETC of unicellular eukaryotes (protists), which are key to understanding the evolution of mitochondria and the ETC. We present an analysis of the ETC proteome from Acanthamoeba castellanii, an ecologically, medically and evolutionarily important member of Amoebozoa (sister to Opisthokonta). Data obtained from tandem mass spectrometric (MS/MS) analyses of purified mitochondria as well as ETC complexes isolated via blue native polyacrylamide gel electrophoresis are combined with the results of bioinformatic queries of sequence databases. Our bioinformatic analyses have identified most of the ETC subunits found in other eukaryotes, confirming and extending previous observations. The assignment of proteins as ETC subunits by MS/MS provides important insights into the primary structures of ETC proteins and makes possible, through the use of sensitive profile-based similarity searches, the identification of novel constituents of the ETC along with the annotation of highly divergent but phylogenetically conserved ETC subunits.

Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

Iodination on tyrosine residues during oxidation with sodium periodate in solid phase extraction of N-linked glycopeptides.

Solid-phase extraction of N-linked glycopeptides (SPEG) using hydrazide-modified supports has become a common sample preparation procedure in glycoproteomic experiments. We demonstrate that iodination of tyrosine residues occur in SPEG as a side reaction during an oxidation step with sodium periodate. MS/MS analysis of oxidized bovine serum albumin and carbonic anhydrase digests revealed a characteristic shift of m/z 125.9 on all y and b fragment ions containing the modified tyrosine residues. Selected reaction monitoring (SRM) measurements showed that the peak intensity from of the iodinated peptides increased during the course of oxidation. After an hour of oxidation, SRM analysis revealed that the strongest signal from an iodinated peptide was approximately one-tenth of the intensity of the corresponding unmodified peptide. Iodinated tyrosine residues were also identified in serum samples subjected to SPEG and analyzed by LC-ESI-MS/MS. We recommend assessing this side reaction by including iodotyrosine as a variable modification when performing database searches on SPEG experiments. For SRM-based acquisitions, we encourage the avoidance of tyrosine-containing glycopeptides or, if this is not practical, monitoring transitions that contain the potential modified iodinated tyrosine residue to monitor the presence of the iodinated form of the glycopeptide.

Targeted comparative proteomics by liquid chromatography/matrix-assisted laser desorption/ionization triple-quadrupole mass spectrometry.

Here we report the first application of a matrix-assisted laser desorption/ionization (MALDI) triple-quadrupole mass spectrometer for targeted proteomics. Employing an amine-specific isotopic labelling approach, the technique was validated using five randomly selected bovine serum albumin peptides differentially labelled at known ratios. An indirect benefit of the isotopic labelling technique is a significant enhancement of the a1 ion in tandem mass (MS/MS) spectra of all peptides studied. Therefore, the a1 ion was selected as the fragment ion for multiple reaction monitoring (MRM) in all cases, eliminating tedious method development and optimization. Accurate quantification was achieved with an average relative standard deviation (RSD) of 5% (n = 5) and a detection limit of 14 amol. The technique was then applied to validate an important virulence biomarker of the fungal pathogen Candida albicans, which was not accurately quantified using global proteomics experiment employing two-dimensional liquid chromatography/electrospray ionization tandem mass spectrometry (2D-LC/ESI)-MS/MS. Using LC/MALDI-MRM analysis of five tryptic peptides, the protein PHR1 was found to be upregulated in the hyphal (pathogenic) form of C. albicans by a factor of 7.7 +/- 0.8.

Proteomic analysis of Candida albicans yeast and hyphal cell wall and associated proteins.

Candida albicans is an important human pathogen that causes systemic infections, predominantly among populations with weakened immune systems. The morphological transition from the yeast to the hyphal state is one of the key factors in C. albicans pathogenesis. Owing to their location at the host-pathogen interface, the cell wall and associated proteins are of interest, especially with respect to the yeast to hyphal transition. This study entailed the proteomic analysis of differentially regulated proteins involved in this transition. The protein profiles of C. albicans DTT/SDS-extractible proteins and the cyanogen bromide (CNBr)/trypsin-extractable proteins of a cell wall-enriched fraction from yeast and hyphae were compared. In total, 107 spots were identified from the DTT/SDS-extractible cell wall-enriched fraction, corresponding to 82 unique proteins. Of these DTT/SDS-extractible proteins, 14 proteins were upregulated and 10 were downregulated in response to hyphal induction. Approximately 6-9% of total cell wall-protein-enriched fraction was found to be resistant to DTT/SDS extraction. Analysis of the DTT/SDS-resistant fraction using a CNBr/trypsin extraction resulted in the identification of 29 proteins. Of these, 17 were identified only in the hyphae, four were identified only in the yeast, and eight were identified in both the yeast and hyphae.

Development of an interleukin-12-deficient mouse model that is permissive for colonization by a motile KE26695 strain of Helicobacter pylori.

The identification of genes associated with colonization and persistence of Helicobacter pylori in the gastric mucosa has been limited by the lack of robust animal models that support infection by strains whose genomes have been completely sequenced. Here we report that an interleukin-12 (IL-12)-deficient mouse (IL-12(-/-) p40 subunit knockout in C57BL/6 mouse) is permissive for infection by a motile variant (KE88-3887) of The Institute For Genomic Research-sequenced strain (KE26695) of H. pylori. The IL-12-deficient mouse was also more permissive for colonization by the mouse-colonizing Sydney 1 strain of H. pylori than were wild-type C57BL/6 mice. Differences in colonization efficiency were demonstrated by mouse challenge with SS1 strains containing loss-of-function mutations in two genes (hspR and hrcA), whose products negatively regulate several heat shock genes. At 5 weeks postinfection, double-knockout mutants (SS1 hspR hrcA) efficiently colonized IL-12-deficient mice (5 of 5 animals compared to 4 of 10 for C57BL6 mice) and bacterial counts were higher in stomachs of IL-12-deficient mice (10(6) versus 10(5) CFU/g of stomach, respectively). IL-12-deficient mice were efficiently colonized by KE88-3887 (29 of 30), but not by nonmotile KE26695, and bacterial numbers (10(4) to 10(5) CFU/g of stomach) were unchanged over an 8-week period postinfection. In contrast, C57BL/6 mice were inefficiently colonized by KE88-3887 (8 of 20 animals with bacterial loads at the limit of detection, approximately 10(3) CFU/g), and infection did not persist much beyond 5 weeks. Cytokine responses (tumor necrosis factor alpha and gamma interferon), pathology, and antral-predominant infection were indistinguishable between IL-12-deficient and C57BL/6 mice. The increased permissiveness of the IL-12-deficient mouse for infection with H. pylori should facilitate whole-genome-based strategies to study genes associated with virulence and immune modulation.