Analysis of plasma proteins using 2D gels and novel fluorescent probes: in search of blood based biomarkers for Alzheimer's disease.

BACKGROUND: The Australian Imaging and Biomarker Lifestyle (AIBL) study of aging is designed to aid the discovery of biomarkers. The current study aimed to discover differentially expressed plasma proteins that could yield a blood-based screening tool for Alzheimer's disease. METHODS: The concentration of proteins in plasma covers a vast range of 12 orders of magnitude. Therefore, to search for medium to low abundant biomarkers and elucidate mechanisms of AD, we immuno-depleted the most abundant plasma proteins and pre-fractionated the remaining proteins by HPLC, prior to two-dimensional gel electrophoresis. The relative levels of approximately 3400 protein species resolved on the 2D gels were compared using in-gel differential analysis with spectrally resolved fluorescent protein detection dyes (Zdyes™). Here we report on analysis of pooled plasma samples from an initial screen of a sex-matched cohort of 72 probable AD patients and 72 healthy controls from the baseline time point of AIBL. RESULTS: We report significant changes in variants of apolipoprotein E, haptoglobin, α1 anti-trypsin, inter-α trypsin inhibitor, histidine-rich glycoprotein, and a protein of unknown identity. α1 anti-trypsin and α1 anti-chymotrypsin demonstrated plasma concentrations that were dependent on APOE ε4 allele dose. Our analysis also identified an association with the level of Vitamin D binding protein fragments and complement factor I with sex. We then conducted a preliminary validation study, on unique individual samples compared to the discovery cohort, using a targeted LC-MS/MS assay on a subset of discovered biomarkers. We found that targets that displayed a high degree of isoform specific changes in the 2D gels were not changed in the targeted MS assay which reports on the total level of the biomarker. CONCLUSIONS: This demonstrates that further development of mass spectrometry assays is needed to capture the isoform complexity that exists in theses biological samples. However, this study indicates that a peripheral protein signature has potential to aid in the characterization of AD.

Identification of C-terminal phosphorylation sites of N-formyl peptide receptor-1 (FPR1) in human blood neutrophils.

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.

Antigen-antibody interface properties: composition, residue interactions, and features of 53 non-redundant structures.

The structures of protein antigen-antibody (Ag-Ab) interfaces contain information about how Ab recognize Ag as well as how Ag are folded to present surfaces for Ag recognition. As such, the Ab surface holds information about Ag folding that resides with the Ab-Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, a data set comprised of 53 non-redundant 3D structures of Ag-Ab complexes was analyzed. We assessed the physical and biochemical features of the Ag-Ab interfaces and the degree to which favored interactions exist between amino acid residues on the corresponding interface surfaces. Amino acid compositional analysis of the interfaces confirmed the dominance of TYR in the Ab paratope-containing surface (PCS), with almost two fold greater abundance than any other residue. Additionally TYR had a much higher than expected presence in the PCS compared to the surface of the whole antibody (defined as the occurrence propensity), along with aromatics PHE, TRP, and to a lesser degree HIS and ILE. In the Ag epitope-containing surface (ECS), there were slightly increased occurrence propensities of TRP and TYR relative to the whole Ag surface, implying an increased significance over the compositionally most abundant LYS>ASN>GLU>ASP>ARG. This examination encompasses a large, diverse set of unique Ag-Ab crystal structures that help explain the biological range and specificity of Ag-Ab interactions. This analysis may also provide a measure of the significance of individual amino acid residues in phage display analysis of Ag binding.

Enhanced sensitivity employing zwitterionic and pI balancing dyes (Z-CyDyes) optimized for 2D-gel electrophoresis based on side chain modifications of CyDye fluorophores. New tools for use in proteomics and diagnostics.

The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents.

Discovery of a Missense Mutation (Q222K) of the APOE Gene from the Australian Imaging, Biomarker and Lifestyle Study.

After age, polymorphisms of the Apolipoprotein E (APOE) gene are the biggest risk factor for the development of Alzheimer's disease (AD). During our investigation to discovery biomarkers in plasma, using 2D gel electrophoresis, we found an individual with and unusual apoE isoelectric point compared to APOE ɛ2, ɛ3, and ɛ4 carriers. Whole exome sequencing of APOE from the donor confirmed a single nucleotide polymorphism (SNP) in exon 4, translating to a rare Q222K missense mutation. The apoE ɛ4 (Q222K) mutation did not form dimers or complexes observed for apoE ɛ2 & ɛ3 proteins.

Proteomic analysis of Sulfolobus solfataricus during Sulfolobus Turreted Icosahedral Virus infection.

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses; however, a major exception are viruses with archaeal hosts. Archaeal virus-host systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homologue in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well-described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.

Proteomic analysis of human epileptic neocortex predicts vascular and glial changes in epileptic regions.

Epilepsy is a common neurological disorder, which is not well understood at the molecular level. Exactly why some brain regions produce epileptic discharges and others do not is not known. Patients who fail to respond to antiseizure medication (refractory epilepsy) can benefit from surgical removal of brain regions to reduce seizure frequency. The tissue removed in these surgeries offers an invaluable resource to uncover the molecular and cellular basis of human epilepsy. Here, we report a proteomic study to determine whether there are common proteomic patterns in human brain regions that produce epileptic discharges. We analyzed human brain samples, as part of the Systems Biology of Epilepsy Project (SBEP). These brain pieces are in vivo electrophysiologically characterized human brain samples withdrawn from the neocortex of six patients with refractory epilepsy. This study is unique in that for each of these six patients the comparison of protein expression was made within the same patient: a more epileptic region was compared to a less epileptic brain region. The amount of epileptic activity was defined for each patient as the frequency of their interictal spikes (electric activity between seizures that is a parameter strongly linked to epilepsy). Proteins were resolved from three subcellular fractions, using a 2D differential gel electrophoresis (2D-DIGE), revealing 31 identified protein spots that changed significantly. Interestingly, glial fibrillary acidic protein (GFAP) was found to be consistently down regulated in high spiking brain tissue and showed a strong negative correlation with spike frequency. We also developed a two-step analysis method to select for protein species that changed frequently among the patients and identified these proteins. A total of 397 protein spots of interest (SOI) were clustered by protein expression patterns across all samples. These clusters were used as markers and this analysis predicted proteomic changes due to both histological differences and molecular pathways, revealed by examination of gene ontology clusters. Our experimental design and proteomic data analysis predicts novel glial changes, increased angiogenesis, and changes in cytoskeleton and neuronal projections between high and low interictal spiking regions. Quantitative histological staining of these same tissues for both the vascular and glial changes confirmed these findings, which provide new insights into the structural and functional basis of neocortical epilepsy.

Metarhodopsin-II stabilization by crosslinked Gtalpha C-terminal peptides and implications for the mechanism of GPCR-G protein coupling.

Metarhodopsin-II is the light-excited form of rhodopsin that triggers G protein function. Metarhodopsin-II is stabilized when the N-terminus of the carboxyl (340-350) tail peptide of the alpha-subunit of transducin (Gtalpha) is crosslinked to rhodopsin cysteine 140 or the 340-350 peptide C-terminus of Gtalpha is crosslinked to rhodopsin cysteine 316. When the N-terminus of the peptide is coupled to C316 the MI<-->MII equilibrium is not affected. The evidence suggests that the N-terminus of the 340-350 region of Gtalpha is located near C140 when transducin stabilizes metarhodopsin-II and alternative explanations are suggested for the effectiveness of the 340-350 Gtalpha tail peptide when bound to C316.

ProMoST (Protein Modification Screening Tool): a web-based tool for mapping protein modifications on two-dimensional gels.

ProMoST is a flexible web tool that calculates the effect of single or multiple posttranslational modifications (PTMs) on protein isoelectric point (pI) and molecular weight and displays the calculated patterns as two-dimensional (2D) gel images. PTMs of proteins control many biological regulatory and signaling mechanisms and 2D gel electrophoresis is able to resolve many PTM-induced isoforms, such as those due to phosphorylation, acetylation, deamination, alkylation, cysteine oxidation or tyrosine nitration. These modifications cause changes in the pI of the protein by adding, removing or changing titratable groups. Proteins differ widely in buffering capacity and pI and therefore the same PTMs may give rise to quite different patterns of pI shifts in different proteins. It is impossible by visual inspection of a pattern of spots on a gel to determine which modifications are most likely to be present. The patterns of PTM shifts for different proteins can be calculated and are often quite distinctive. The theoretical gel images produced by ProMoST can be compared to the experimental 2D gel results to implicate probable PTMs and focus efforts on more detailed study of modified proteins. ProMoST has been implemented as cgi script in Perl available on a WWW server at http://proteomics.mcw.edu/promost.

The Quantum Biology of Reactive Oxygen Species Partitioning Impacts Cellular Bioenergetics.

Quantum biology is the study of quantum effects on biochemical mechanisms and biological function. We show that the biological production of reactive oxygen species (ROS) in live cells can be influenced by coherent electron spin dynamics, providing a new example of quantum biology in cellular regulation. ROS partitioning appears to be mediated during the activation of molecular oxygen (O2) by reduced flavoenzymes, forming spin-correlated radical pairs (RPs). We find that oscillating magnetic fields at Zeeman resonance alter relative yields of cellular superoxide (O2•-) and hydrogen peroxide (H2O2) ROS products, indicating coherent singlet-triplet mixing at the point of ROS formation. Furthermore, the orientation-dependence of magnetic stimulation, which leads to specific changes in ROS levels, increases either mitochondrial respiration and glycolysis rates. Our results reveal quantum effects in live cell cultures that bridge atomic and cellular levels by connecting ROS partitioning to cellular bioenergetics.

Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints.

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.

A new method for mapping discontinuous antibody epitopes to reveal structural features of proteins.

Antibodies that bind to protein surfaces of interest can be used to report the three-dimensional structure of the protein as follows: Proteins are composed of linear polypeptide chains that fold together in complex spatial patterns to create the native protein structure. These folded structures form binding sites for antibodies. Antibody binding sites are typically "assembled" on the protein surface from segments that are far apart in the primary amino acid sequence of the target proteins. Short amino acid probe sequences that bind to the active region of each antibody can be used as witnesses to the antibody epitope surface and these probes can be efficiently selected from random sequence peptide libraries. This paper presents a new method to align these antibody epitopes to discontinuous regions of the one-dimensional amino acid sequence of a target protein. Such alignments of the epitopes indicate how segments of the protein sequence must be folded together in space and thus provide long-range constraints for solving the 3-D protein structure. This new antibody-based approach is applicable to the large fraction of proteins that are refractory to current approaches for structure determination and has the additional advantage of requiring very small amounts of the target protein. The binding site of an antibody is a surface, not just a continuous linear sequence, so the epitope mapping alignment problem is outside the scope of classical string alignment algorithms, such as Smith-Waterman. We formalize the alignment problem that is at the heart of this new approach, prove that the epitope mapping alignment problem is NP-complete, and give some initial results using a branch-and-bound algorithm to map two real-life cases. Initial results for two validation cases are presented for a graph-based protein surface neighbor mapping procedure that promises to provide additional spatial proximity information for the amino acid residues on the protein surface.

Proteomic and systems biology analysis of the monocyte response to Coxiella burnetii infection.

Coxiella burnetii is an obligate intracellular bacterial pathogen and the causative agent of Q fever. Chronic Q fever can produce debilitating fatigue and C. burnetii is considered a significant bioterror threat. C. burnetii occupies the monocyte phagolysosome and although prior work has explained features of the host-pathogen interaction, many aspects are still poorly understood. We have conducted a proteomic investigation of human Monomac I cells infected with the Nine Mile Phase II strain of C. burnetii and used the results as a framework for a systems biology model of the host response. Our principal methodology was multiplex differential 2D gel electrophoresis using ZDyes, a new generation of covalently linked fluorescent protein detection dyes under development at Montana State University. The 2D gel analysis facilitated the detection of changes in posttranslational modifications on intact proteins in response to infection. The systems model created from our data a framework for the design of experiments to seek a deeper understanding of the host-pathogen interactions.

Proteomic and systems biology analysis of monocytes exposed to securinine, a GABA(A) receptor antagonist and immune adjuvant.

Securinine, a GABA(A) receptor antagonist, has been reported to enhance monocyte cell killing of Coxiella burnetii without obvious adverse effects in vivo. We employed multiplex 2D gel electrophoresis using Zdyes, a new generation of covalently linked fluorescent differential protein detection dyes to analyze changes in the monocyte proteome in response to Securinine. Securinine antagonism of GABA(A) receptors triggers the activation of p38. We used the differential protein expression results to guide a search of the literature and network analysis software to construct a systems biology model of the effect of Securinine on monocytes. The model suggests that various metabolic modulators (fatty acid binding protein 5, inosine 5'-monophosphate dehydrogenase, and thioredoxin) are at least partially reshaping the metabolic landscape within the monocytes. The actin bundling protein L-plastin, and the Ca(2+) binding protein S100A4 also appear to have important roles in the immune response stimulated by Securinine. Fatty acid binding protein 5 (FABP5) may be involved in effecting lipid raft composition, inflammation, and hormonal regulation of monocytes, and the model suggests that FABP5 may be a central regulator of metabolism in activated monocytes. The model also suggests that the heat shock proteins have a significant impact on the monocyte immune response. The model provides a framework to guide future investigations into the mechanisms of Securinine action and with elaboration may help guide development of new types of immune adjuvants.

Global analysis of viral infection in an archaeal model system.

The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal virus-host systems of particular interest because the hosts generally root near the base of phylogenetic trees, while some of the viruses have clear structural similarities to those that infect prokaryotic and eukaryotic cells. Despite the advantageous position for use in evolutionary studies, little is known about archaeal viruses or how they interact with their hosts, compared to viruses of bacteria and eukaryotes. In addition, many archaeal viruses have been isolated from extreme environments and present a unique opportunity for elucidating factors that are important for existence at the extremes. In this article we focus on virus-host interactions using a proteomics approach to study Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2. Using cultures grown from the ATCC cell stock, a single cycle of STIV infection was sampled six times over a 72 h period. More than 700 proteins were identified throughout the course of the experiments. Seventy one host proteins were found to change their concentration by nearly twofold (p < 0.05) with 40 becoming more abundant and 31 less abundant. The modulated proteins represent 30 different cell pathways and 14 clusters of orthologous groups. 2D gel analysis showed that changes in post-translational modifications were a common feature of the affected proteins. The results from these studies showed that the prokaryotic antiviral adaptive immune system CRISPR-associated proteins (CAS proteins) were regulated in response to the virus infection. It was found that regulated proteins come from mRNAs with a shorter than average half-life. In addition, activity-based protein profiling (ABPP) profiling on 2D-gels showed caspase, hydrolase, and tyrosine phosphatase enzyme activity labeling at the protein isoform level. Together, this data provides a more detailed global view of archaeal cellular responses to viral infection, demonstrates the power of quantitative two-dimensional differential gel electrophoresis and ABPP using 2D gel compatible fluorescent dyes.

Invariant local conformation in p22phox p.Y72H polymorphisms suggested by mass spectral analysis of crosslinked human neutrophil flavocytochrome b.

The NADPH oxidase of phagocytic leukocytes generates superoxide that plays a critical role in innate immunity and inflammatory responses. The integral membrane protein flavocytochrome b (Cyt b, a.k.a. cytochrome b(558/559)) is the catalytic core of the complex and serves as a prototype for homologs important in regulating signaling networks in a wide variety of animal and plant cells. Our analysis identifies a naturally-occurring Tyr72/His72 polymorphism (p.Y72H) in the p22(phox) subunit of Cyt b at the protein level that has been recognized at the nucleotide level (c.214T > C, formerly C242T) and implicated in cardiovascular disease. In the present study, Cyt b was isolated from human neutrophils and reacted with chemical crosslinkers for subsequent structure analysis by MALDI mass spectrometry. Following mild chemical modification of Cyt b with two pairs of isotopically-differentiated lysine crosslinkers: BS(2)G-d(0)/d(4) and BS(3)-d(0)/d(4), the reaction mixtures were digested with trypsin and purified on C(18)ZipTips to generate samples for mass analysis. MALDI analysis of tryptic digests from each of the above reactions revealed a series of masses that could be assigned to p22(phox) residues 68-85, assuming an intra-molecular crosslink between Lys71 and Lys78. In addition to the 30 ppm mass accuracy obtained with internal mass calibration, increased confidence in the assignment of the crosslinks was provided by the presence of the diagnostic mass patterns resulting from the isotopically-differentiated crosslinking reagent pairs and the Tyr72/His72 p22(phox) polymorphisms in the crosslinked peptides. This work identifies a novel, low-resolution distance constraint in p22(phox) and suggests that the medically-relevant p.Y72H polymorphism has an invariant structural motif in this region. Because position 72 in p22(phox) lies outside regions identified as interactive with other oxidase components, the structural invariance also provides additional support for maturational differences as the source of the wide variation in observed reactive oxygen species production by cells expressing p.Y72H.

Facile fingerstick insulin analysis: Application to monitoring postprandial insulin responses to snack foods.

BACKGROUND: Energy intake from snacks has been increasing in the American diet, but insulin and glucose responses to foods are generally reported for meal-sized portions (800-1200 kJ). Established methods for insulin determination routinely use indwelling catheters and radioimmunoassay (RIA). The aim of the present study was to develop a more facile method, collecting fingerstick blood samples and measuring insulin with precise ELISA, and then applying this method to determine responses to snack-sized food portions. METHODS: Six healthy, fasting adult volunteers consumed seven different snack foods on separate days, containing approximately 400 kJ/portion. Insulin was measured by ELISA and glucose was measured with the hexokinase procedure in samples collected by fingerstick at 0, 30, and 60 min after consumption of the snack food. RESULTS: A portion of doughnut (half a glazed doughnut) led to marked changes in insulin and glucose; skim milk, an apple, and oatmeal changed insulin significantly; wrinkled peas resulted in a lower glucose response than smooth peas; and walnuts led to non-significant changes in both insulin and glucose over a 60-min period. CONCLUSIONS: The fingerstick sampling and insulin measurement procedure is simple, economical, and more precise than established RIA. The method can be applied to children and adults to monitor insulin responses following food consumption, as well as during therapeutic assessments or intervention trials. Public health advisories regarding snacks that minimize increases in insulin are desirable for individuals trying to reduce or maintain their weight, because elevated insulin stimulates carbohydrate conversion to fat and suppresses the mobilization of stored triglycerides for energy generation.

Elevating optimal human nutrition to a central goal of plant breeding and production of plant-based foods.

High-yielding cereals and other staples have produced adequate calories to ward off starvation for much of the world over several decades. However, deficiencies in certain amino acids, minerals, vitamins and fatty acids in staple crops, and animal diets derived from them, have aggravated the problem of malnutrition and the increasing incidence of certain chronic diseases in nominally well-nourished people (the so-called diseases of civilization). Enhanced global nutrition has great potential to reduce acute and chronic disease, the need for health care, the cost of health care, and to increase educational attainment, economic productivity and the quality of life. However, nutrition is currently not an important driver of most plant breeding efforts, and there are only a few well-known efforts to breed crops that are adapted to the needs of optimal human nutrition. Technological tools are available to greatly enhance the nutritional value of our staple crops. However, enhanced nutrition in major crops might only be achieved if nutritional traits are introduced in tandem with important agronomic yield drivers, such as resistance to emerging pests or diseases, to drought and salinity, to herbicides, parasitic plants, frost or heat. In this way we might circumvent a natural tendency for high yield and low production cost to effectively select against the best human nutrition. Here we discuss the need and means for agriculture, food processing, food transport, sociology, nutrition and medicine to be integrated into new approaches to food production with optimal human nutrition as a principle goal.

C-terminal tail phosphorylation of N-formyl peptide receptor: differential recognition of two neutrophil chemoattractant receptors by monoclonal antibodies NFPR1 and NFPR2.

The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni(2+)-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45-75 kDa species (peak M(r) approximately 60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of M(r) approximately 40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting approximately 60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337-346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils.

Proteomic mapping of the hyperthermophilic and acidophilic archaeon Sulfolobus solfataricus P2.

A proteomic map of Sulfolobus solfataricus P2, an archaeon that grows optimally at 80 degrees C and pH 3.2, was developed using high-resolution 2-DE and peptide mass fingerprinting. A total of 867 protein spots (659 aqueous Tris-soluble spots and 208 aqueous Tris-insoluble) were mapped over IPG 3-10, 4-7, and 6-11, with second-dimensional gels made of 8-18% polyacrylamide. Three hundred and twenty-four different gene products were represented by the 867 spots, with 274 gene products being identified in the Tris-soluble fractions and 100 gene products in the Tris-insoluble portion. Fifty gene products were found on gels from both fractions. Additionally, an average of 1.50 +/- 0.12 isoforms/protein was identified. This mapping study confirmed the expression of proteins involved in numerous metabolic, transport, energy production, nucleic acid replication, translation, and transcription pathways. Of particular interest, phosphoenolpyruvate carboxykinase (SSO2537) was detected even though the pathway for gluconeogenesis is unknown for this archaeon. Tris-soluble fractions contained many cytosolic proteins while Tris-insoluble fractions contained many membrane-associated proteins, including ABC transporters and an ATP synthase. This study provides an optimized 2-DE approach for investigating the biochemical pathways and post-translational modifications employed by Sulfolobus to survive in its extreme environment.