Morphological and proteomic analysis of biofilms from the Antarctic archaeon, Halorubrum lacusprofundi.

Biofilms enhance rates of gene exchange, access to specific nutrients, and cell survivability. Haloarchaea in Deep Lake, Antarctica, are characterized by high rates of intergenera gene exchange, metabolic specialization that promotes niche adaptation, and are exposed to high levels of UV-irradiation in summer. Halorubrum lacusprofundi from Deep Lake has previously been reported to form biofilms. Here we defined growth conditions that promoted the formation of biofilms and used microscopy and enzymatic digestion of extracellular material to characterize biofilm structures. Extracellular DNA was found to be critical to biofilms, with cell surface proteins and quorum sensing also implicated in biofilm formation. Quantitative proteomics was used to define pathways and cellular processes involved in forming biofilms; these included enhanced purine synthesis and specific cell surface proteins involved in DNA metabolism; post-translational modification of cell surface proteins; specific pathways of carbon metabolism involving acetyl-CoA; and specific responses to oxidative stress. The study provides a new level of understanding about the molecular mechanisms involved in biofilm formation of this important member of the Deep Lake community.

Developing a genetic manipulation system for the Antarctic archaeon, Halorubrum lacusprofundi: investigating acetamidase gene function.

No systems have been reported for genetic manipulation of cold-adapted Archaea. Halorubrum lacusprofundi is an important member of Deep Lake, Antarctica (~10% of the population), and is amendable to laboratory cultivation. Here we report the development of a shuttle-vector and targeted gene-knockout system for this species. To investigate the function of acetamidase/formamidase genes, a class of genes not experimentally studied in Archaea, the acetamidase gene, amd3, was disrupted. The wild-type grew on acetamide as a sole source of carbon and nitrogen, but the mutant did not. Acetamidase/formamidase genes were found to form three distinct clades within a broad distribution of Archaea and Bacteria. Genes were present within lineages characterized by aerobic growth in low nutrient environments (e.g. haloarchaea, Starkeya) but absent from lineages containing anaerobes or facultative anaerobes (e.g. methanogens, Epsilonproteobacteria) or parasites of animals and plants (e.g. Chlamydiae). While acetamide is not a well characterized natural substrate, the build-up of plastic pollutants in the environment provides a potential source of introduced acetamide. In view of the extent and pattern of distribution of acetamidase/formamidase sequences within Archaea and Bacteria, we speculate that acetamide from plastics may promote the selection of amd/fmd genes in an increasing number of environmental microorganisms.

Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System.

Sirtuin proteins have a variety of intracellular targets, thereby regulating multiple biological pathways including neurodegeneration. However, relatively little is currently known about the role or expression of the 7 mammalian sirtuins in the central nervous system. Western blotting, PCR and ELISA are the main techniques currently used to measure sirtuin levels. To achieve sufficient sensitivity and selectivity in a multiplex-format, a targeted mass spectrometric assay was developed and validated for the quantification of all seven mammalian sirtuins (SIRT1-7). Quantification of all peptides was by multiple reaction monitoring (MRM) using three mass transitions per protein-specific peptide, two specific peptides for each sirtuin and a stable isotope labelled internal standard. The assay was applied to a variety of samples including cultured brain cells, mammalian brain tissue, CSF and plasma. All sirtuin peptides were detected in the human brain, with SIRT2 being the most abundant. Sirtuins were also detected in human CSF and plasma, and guinea pig and mouse tissues. In conclusion, we have successfully applied MRM mass spectrometry for the detection and quantification of sirtuin proteins in the central nervous system, paving the way for more quantitative and functional studies.

Phenotypic Characterization of Insulin-Resistant and Insulin-Sensitive Obesity.

CONTEXT: Whereas insulin resistance and obesity coexist, some obese individuals remain insulin sensitive. OBJECTIVE: We examined phenotypic and metabolic factors associated with insulin sensitivity in both muscle and liver in obese individuals. DESIGN AND PARTICIPANTS: Sixty-four nondiabetic obese adults (29 males) underwent hyperinsulinemic (15 and 80 mU/m(2) · min)-euglycemic clamps with deuterated glucose. Top tertile subjects for glucose infusion rate during the high-dose insulin clamp were assigned Musclesen and those in the lower two tertiles were assigned Muscleres. Secondarily, top tertile subjects for endogenous glucose production suppression during the low-dose insulin clamp were deemed Liversen and the remainder Liverres. MAIN OUTCOMES MEASURES: Clinical and laboratory parameters and visceral, subcutaneous, liver, and pancreatic fat were compared. RESULTS: Musclesen and Muscleres had similar body mass index and total fat (P > .16), but Musclesen had lower glycated hemoglobin (P < .001) and systolic (P = .01) and diastolic (P = .03) blood pressure (BP). Despite similar sc fat (P = 1), Musclesen had lower visceral (P < .001) and liver (P < .001) fat. Liversen had lower visceral (P < .01) and liver (P < .01) fat and C-reactive protein (P = .02) than Liverres. When subjects were grouped by both glucose infusion rate during the high-dose insulin clamp and endogenous glucose production suppression, insulin sensitivity at either muscle or liver conferred apparent protection from the adverse metabolic features that characterized subjects insulin resistant at both sites. High-density lipoprotein-cholesterol, 1-hour glucose, systolic BP, and triglycerides explained 54% of the variance in muscle insulin sensitivity. CONCLUSIONS: Obese subjects who were insulin sensitive at muscle and/or liver exhibited favorable metabolic features, including lower BP, liver and visceral adiposity. This study identifies factors associated with, and possibly contributing to, insulin sensitivity in obesity.

Quantitative proteomics of delirium cerebrospinal fluid.

Delirium is a common cause and complication of hospitalization in older people, being associated with higher risk of future dementia and progression of existing dementia. However relatively little data are available on which biochemical pathways are dysregulated in the brain during delirium episodes, whether there are protein expression changes common among delirium subjects and whether there are any changes which correlate with the severity of delirium. We now present the first proteomic analysis of delirium cerebrospinal fluid (CSF), and one of few studies exploring protein expression changes in delirium. More than 270 proteins were identified in two delirium cohorts, 16 of which were dysregulated in at least 8 of 17 delirium subjects compared with a mild Alzheimer's disease neurological control group, and 31 proteins were significantly correlated with cognitive scores (mini-mental state exam and acute physiology and chronic health evaluation III). Bioinformatics analyses revealed expression changes in several protein family groups, including apolipoproteins, secretogranins/chromogranins, clotting/fibrinolysis factors, serine protease inhibitors and acute-phase response elements. These data not only provide confirmatory evidence that the inflammatory response is a component of delirium, but also reveal dysregulation of protein expression in a number of novel and unexpected clusters of proteins, in particular the granins. Another surprising outcome of this work is the level of similarity of CSF protein profiles in delirium patients, given the diversity of causes of this syndrome. These data provide additional elements for consideration in the pathophysiology of delirium as well as potential biomarker candidates for delirium diagnosis.

Green fluorescent protein expression triggers proteome changes in breast cancer cells.

Green fluorescent protein (GFP) is the most commonly used reporter of expression in cell biology despite evidence that it affects the cell physiology. The molecular mechanism of GFP-associated modifications has been largely unexplored. In this paper we investigated the proteome modifications following stable expression of GFP in breast cancer cells (MDA-MB-231). A combination of three different proteome analysis methods (2-DE, iTRAQ, label-free) was used to maximise proteome coverage. We found that GFP expression induces changes in expression of proteins that are associated with protein folding, cytoskeletal organisation and cellular immune response. In view of these findings, the use of GFP as a cell reporter should be carefully monitored.

The role of polyphenols in the modulation of sirtuins and other pathways involved in Alzheimer's disease.

Alzheimer's disease (AD) is characterised by extracellular amyloid deposits, neurofibrillary tangles, synaptic loss, inflammation and extensive oxidative stress. Polyphenols, which include resveratrol, epigallocatechin gallate and curcumin, have gained considerable interest for their ability to reduce these hallmarks of disease and their potential to slow down cognitive decline. Although their antioxidant and free radical scavenging properties are well established, more recently polyphenols have been shown to produce other important effects including anti-amyloidogenic activity, cell signalling modulation, effects on telomere length and modulation of the sirtuin proteins. Brain accessible polyphenols with multiple effects on pathways involved in neurodegeneration and ageing may therefore prove efficacious in the treatment of age-related diseases such as AD, although the evidence for this so far is limited. This review aims to explore the known effects of polyphenols from various natural and synthetic sources on brain ageing and neurodegeneration, and to examine their multiple mechanisms of action, with an emphasis on the role that the sirtuin pathway may play and the implications this may have for the treatment of AD.

Proteomic assessment of host-associated microevolution in the fungus Thielaviopsis basicola.

Thielaviopsis basicola, a soil-borne pathogen with a broad host range and a cosmopolitan distribution, is emerging as a major risk to sustainable cotton production in Australia. Previous studies suggested that host specialization has occurred making T. basicola an ideal model for a comparative proteomic analysis of strains isolated from different hosts. Elucidation of the genomic diversity and investigation of the functional differences in the Australian population could provide valuable information towards disease control. In this study, isolates of T. basicola were investigated for genomic (internal transcribed spacers region), proteomic and cotton virulence level variations. Internal transcribed spacers sequence analysis revealed that isolates are grouped based on host of origin irrespective of geographical origin. At the proteome level a degree of diversity was apparent and hierarchical clustering analysis of the data also demonstrated a close correlation between the proteome and the host of origin. LC-MS/MS analysis and identification using cross-species similarity searching and de novo sequencing of host-specific differentially expressed proteins and the virulence-correlated proteome allowed successful identification of 43 spots. The majority were found to be involved in metabolism. Spots that were correlated with host and virulence differences included a hypothetical protein with a Rossman-fold NAD(P)(+)-binding protein domain, glyceraldehyde-3-phosphate dehydrogenase, arginase and tetrahydroxynaphthalene reductase.

Improved activity and stability of alkaline phosphatases from psychrophilic and mesophilic organisms by chemically modifying aliphatic or amino groups using tetracarboxy-benzophenone derivatives.

The activity-stability-structure relationship of the cold-active alkaline phosphatase from Red Arctic shrimp, Pandalus borealis (SAP) was studied by chemically modifying aliphatic (C-H) or amino (NH2) groups using benzophenone tetracarboxylic derivatives in either a light (UV-A) or dark reaction. The response of the cold-adapted enzyme was compared to a similarly modified calf alkaline phosphatase (CAP). MALDI-TOF-MS was used to determine the extent and nature of the modifications in both SAP and CAP. On average 2 to 4 amino acid residues were linked to a BP-modifier, with up to 18 to 21 amino acids modified in a smaller portion of the material. The effect of the modifications on kinetic and thermodynamic properties varied with the enzyme and type of modification. The aliphatic-group modified SAP demonstrated typical characteristics of a mesophilic enzyme, consistent with an activity-stability trade-off where gain in thermostability was attained at the expense of decreased activity. In contrast, the activity of the amino-group modified SAP attained an even more psychrophilic character with respect to its kinetic (increase in kcat and Km) and thermodynamic (reduction in deltaH#) properties. Interestingly, the amino-group modified SAP also acquired higher thermostability, thus demonstrating that both activity and stability can be simultaneously enhanced using chemical modification. The study demonstrates the applicability of benzophenone chemical modification for improving the thermal properties of enzymes from psychrophiles and mesophiles.

Forms that Inform.

OBJECTIVES: Sound medical decisions are easier for clinicians who have essential patient data in the right place at the right time. Our goal was to develop a usable form to guide clinical decisions and then test it using actual cases. METHODS: The authors designed a form to represent data from patients with chest pain; it was revised several times. We incorporated opinions from clinician-users as well as evidence from the literature to improve usability. To test the design, we filled out forms with actual patient data derived from Emergency Department charts of patients who presented with chest pain. We then validated the design by having house officer reviewers make the decision to admit, observe in the ER for one day, or discharge subjects based entirely on a one-page form. RESULTS: Thirty-three house officers reviewed our initial design and made suggestions. Our literature search yielded a number of factors discriminating ischemic from non-ischemic chest pain. Sixteen factors were included on a finalized form in the rank order assigned by reviewing physicians. Over 4 days, data from 29 subjects were used to fill out copies of the form. Based purely on the completed forms, house officers made decisions to admit, discharge, or observe all 29 subjects in less than 30 minutes. CONCLUSIONS: Forms have traditionally been employed to record and organize data. Here we show how principles of usability engineering can be used to create a form to meet the needs of users and even encourage evidence-based practice.

Matrix-assisted laser-desorption time-of flight ionisation and high-performance liquid chromatography-electrospray ionisation mass spectral analyses of two glycosylated recombinant epoetins.

Mass spectrometric analyses of the recombinant proteins in Eprex and Aranesp were undertaken with the goal of producing reference mass spectra and evaluating strategies to improve its applicability as a method for equine and canine doping control of these substances. A simple, low chemical noise deglycosylation reaction removed microheterogeneity due to post-translational carbohydrate attachment and both proteins were detectable using MALDI-TOF-MS. Deglycosylated human erythropoietin (hEPO) was also detected using HPLC-ESI-MS. This is the first time that spectra of deglycosylated Eprex and Aranesp have been published. Eight synthetic reference standards, which match peptides produced by endoproteinase Glu-C enzymatic cleavage of Aranesp and/or Eprex, were analysed by ESI-MS and ESI-MS-MS. The E12 Glu-C peptide, common to both proteins, was detected at the low femtomole-level using gradient nano-HPLC-ESI-MS-MS in the positive ion mode.

Measurements of protein carbonyls, ortho- and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats.

Mitochondrial bioenergetic function is often reported to decline with age and the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of mitochondrial oxidative damage in aging. We hypothesized that, as radical production in mitochondria is greater than the rest of the cell, protein oxidative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hypotheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta-tyrosine levels in protein hydrolysates were measured by a gas chromatography/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxidative damage in mitochondria relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondrial proteins on aging. Mitochondrial oxidative phosphorylation complex activities were also assessed revealing 39-42% decreases in F0F1--ATP synthase activity in liver and heart on aging, but not in other oxidative phosphorylation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging.

Fluorometric and mass spectrometric analysis of nonenzymatic glycosylated albumin.

Albumin is the major transport protein in blood and intramolecular movement contributes to this function. Nonenzymatic glycosylation (NEG) of albumin occurs in diabetes and, in this study, fluorometric methods were used to determine the effect of increasing levels of NEG upon intramolecular movement in human serum albumin. Low levels of NEG significantly reduced and left-shifted Trp fluorescence, reduced quenching by acrylamide and inhibited penetration of bis-ANS, while these changes became only modestly more pronounced at higher levels of NEG. Mass spectrometry of tryptic and CNBr NEG-HSA fragments identified potential glycosylation sites and demonstrated only late glycosylation of the C- and N-terminal regions of the protein. Similar changes in diabetes may contribute to altered transport function in these patients.

Amino Acid analysis of peptides and proteins on the femtomole scale by gas chromatography/mass spectrometry.

Amino acid analysis (AAA) is a useful aid in protein chemistry, but its routine application is limited by a modest limit of detection. Typically, 10 pmol of material is required, but even at this level the reproducibility can be poor. We have employed isotope dilution gas chromatography electron capture negative ionization mass spectrometry (GC/ECNI/MS) to provide accurate and reliable data on less than 100 fmol of material. Precision and accuracy are good, and all 20 non-hydrolyzed amino acids can be determined in this manner. The protein is hydrolyzed (HCl), and then a cocktail, composed of all 20 amino acids as stable isotope-labeled forms (i.e., (13)C and (2)H), is added. The mixture of protein-derived and stable isotope-labeled amino acids is then converted to volatile electron-capturing derivatives with a multistep approach employing heptafluorobutyric anhydride (HFBA), pentafluorobenzyl bromide (PFBBr), and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). These derivatives are then injected directly into the GC/MS system. Groups of selected ions, characteristic of each derivatized amino acid, are thereafter monitored at appropriate time intervals. The ratios of the ion current for the selected ions for the native amino acid and its labeled form are determined and converted to absolute amounts of the native amino acids in the protein hydrolyzate by reference to standard samples prepared at the time of the analysis.

Mass spectrometric identification and quantification of hemorphins extracted from human adrenal and pheochromocytoma tissue.

The hemorphins are a family of recently identified opioid receptor binding peptides derived from the proteolytic processing of the beta, gamma, delta, and epsilon chains of hemoglobin. They have previously been identified at high concentration in human pituitary glands and in the CSF of patients with cerebral bleeding. Hemorphins are potent inhibitors of angiotensin converting enzyme and therefore possibly have a role to play in blood pressure regulation. We report the presence of four hemorphin peptides in extracts of normal adrenal tissue and in pheochromocytoma tumors. The hemorphins were quantified and structurally characterized using mass spectrometry. High concentrations of hemorphins were found in all samples, comparable with the levels reported in the literature for pituitary and brain tissue.

Conserved motifs as the basis for recognition of homologous proteins across species boundaries using peptide-mass fingerprinting.

Two-dimensional gel electrophoresis of any biological system presently resolves a plethora of highly purified proteins for which no function or identity has been determined. Theoretical and experimental data were used to demonstrate that peptide-mass fingerprinting (PMF) could aid in the recognition of conserved motifs across species boundaries, and thereby assist in attributing putative function to some of these molecules. Amino acids residue substitutions produced by biological diversity and phylogenetic distance combine to highlight regions of functional significance within proteins. Using 10 prokaryotic and two eukaryotic elongation factors (EF), up to 25 peptide fragments (> 800 Da) per molecule were compared across species boundaries within a 12 x 12 contingency table (66 cross-species comparisons), based upon the degree of molecular mass and amino acid sequence identity. Total amino acid sequence identity ranged from 29.4-80.9% for these molecules. Peptide fragments with homologous sequence across three or more EF were defined as containing, or being near to, conserved functional motifs. Twelve such fragments (> 800 Da) were found in this group of proteins. In addition, an 808.9 Da peptide of unknown functional significance was seen to occur in three of the 12 molecules studied and in another three EF-Tu molecules. At the 83% (five of six residues) identity level, this fragment was found in a further 35 EF-Tu molecules and in 14 unrelated proteins. Further investigation should reveal a role for this fragment (motif) in structural integrity or protein function. A FASTA search conducted on a peptide fragment containing a conserved GTP-binding motif (GHVDHGK) of EF-Tu from Euglena gracilis was used as an example to putatively attribute partial function to three hypothetical proteins derived from DNA sequencing initiatives.

Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium.

A protein map of the smallest known self-replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two-dimensional gel electrophoresis. A further 19 proteins were subjected to peptide-mass fingerprinting using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and 4 were subjected to N-terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide-mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of 'hierarchical' analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or 'proteome' is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.

Cross-species identification of proteins separated by two-dimensional gel electrophoresis using matrix-assisted laser desorption ionisation/time-of-flight mass spectrometry and amino acid composition.

Amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry were used to identify nine of twelve proteins originally separated by two-dimensional electrophoresis and derived from an organism poorly defined at the molecular level (Spiroplasma melliferum). Two of three unidentified proteins appeared to be novel. The percentage amino acid composition and the molecular mass of peptide fragments generated by tryptic digestion were used to search the PIR/SWISS-PROT and MOWSE databases respectively. Lists of candidate proteins were independently generated and ranked from data obtained by both methods. A putative identification was allocated when a single candidate protein appeared in both lists of computer-generated rankings. Results were verified using N-terminal protein microsequencing. The combined use of amino acid composition and MALDI-TOF mass spectrometry allowed a high degree of confidence to be placed in such identifications because they were based upon homologous data sets of at least 20 parameters (16 amino acids and 4-10 tryptic digest fragments). A further two parameters, estimated M(r) and, to a lesser extent, pI, were also used to reinforce this measure of confidence. Ranking of candidate proteins by one method alone could lead to false identification. Both techniques can process large numbers of samples rapidly. In light of the increasing number of entries in both gene and protein databases, this approach is likely to become an essential first step for the characterisation of proteins, particularly across species boundaries.

Quantitative analysis of low molecular weight compounds of biological interest by matrix-assisted laser desorption ionization.

Internal standards were used to demonstrate that matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can be applied to the quantitative analysis of low molecular weight polar compounds. Three examples were tested: a standard curve for 3,4-dihydroxyphenylalanine (DOPA) was prepared using a stable isotope analogue (i.e., [13C6]DOPA) as an internal standard; [2H16]-acetylcholine was employed as an internal standard for the quantification of acetylcholine; and in the final example, the peptide Ac-Ser-Ile-Arg-His-Tyr-NH2 was used as an internal standard for the quantification of the peptide H-Ser-Ala-Leu-Arg-His-Tyr-NH2. In each instance, straight line fits (r2 > 0.95) demonstrate that MALDI is a viable approach for the quantitative analysis of low molecular weight analytes.