Prebiotic Membranes and Micelles Do Not Inhibit Peptide Formation During Dehydration.

Cycles of dehydration and rehydration could have enabled formation of peptides and RNA in otherwise unfavorable conditions on the early Earth. Development of the first protocells would have hinged upon colocalization of these biopolymers with fatty acid membranes. Using atomic force microscopy, we find that a prebiotic fatty acid (decanoic acid) forms stacks of membranes after dehydration. Using LC-MS-MS (liquid chromatography-tandem mass spectrometry) with isotope internal standards, we measure the rate of formation of serine dipeptides. We find that dipeptides form during dehydration at moderate temperatures (55 °C) at least as fast in the presence of decanoic acid membranes as in the absence of membranes. Our results are consistent with the hypothesis that protocells could have formed within evaporating environments on the early Earth.

De novo design of tyrosine and serine kinase-driven protein switches.

Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.

Staging Encystation Progression in Giardia lamblia Using Encystation-Specific Vesicle Morphology and Associating Molecular Markers.

Differentiation into environmentally resistant cysts is required for transmission of the ubiquitous intestinal parasite Giardia lamblia. Encystation in Giardia requires the production, processing and transport of Cyst Wall Proteins (CWPs) in developmentally induced, Golgi-like, Encystation Specific Vesicles (ESVs). Progress through this trafficking pathway can be followed by tracking CWP localization over time. However, there is no recognized system to distinguish the advancing stages of this process which can complete at variable rates depending on how encystation is induced. Here, we propose a staging system for encysting Giardia based on the morphology of CWP1-stained ESVs. We demonstrate the molecular distinctiveness of maturing ESVs at these stages by following GlRab GTPases through encystation. Previously, we established that Giardia's sole Rho family GTPase, GlRac, associates with ESVs and has a role in regulating their maturation and the secretion of their cargo. As a proof of principle, we delineate the relationship between GlRac and ESV stages. Through proteomic studies, we identify putative interactors of GlRac that could be used as additional ESV stage markers. This staging system provides a common descriptor of ESV maturation regardless of the source of encysting cells. Furthermore, the identified set of molecular markers for ESV stages will be a powerful tool for characterizing trafficking mutants that impair ESV maturation and morphology.

Assessing Protein Sequence Database Suitability Using De Novo Sequencing.

The analysis of samples from unsequenced and/or understudied species as well as samples where the proteome is derived from multiple organisms poses two key questions. The first is whether the proteomic data obtained from an unusual sample type even contains peptide tandem mass spectra. The second question is whether an appropriate protein sequence database is available for proteomic searches. We describe the use of automated de novo sequencing for evaluating both the quality of a collection of tandem mass spectra and the suitability of a given protein sequence database for searching that data. Applications of this method include the proteome analysis of closely related species, metaproteomics, and proteomics of extinct organisms.

TCP4-dependent induction of CONSTANS transcription requires GIGANTEA in photoperiodic flowering in Arabidopsis.

Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.

Model System-Guided Protein Interaction Mapping for Virus Isolated from Phloem Tissue.

Phloem localization of plant viruses is advantageous for acquisition by sap-sucking vectors but hampers host-virus protein interaction studies. In this study, Potato leafroll virus (PLRV)-host protein complexes were isolated from systemically infected potato, a natural host of the virus. Comparing two different co-immunoprecipitation (co-IP) support matrices coupled to mass spectrometry (MS), we identified 44 potato proteins and one viral protein (P1) specifically associated with virus isolated from infected phloem. An additional 142 proteins interact in complex with virus at varying degrees of confidence. Greater than 80% of these proteins were previously found to form high confidence interactions with PLRV isolated from the model host Nicotiana benthamiana. Bioinformatics revealed that these proteins are enriched for functions related to plasmodesmata, organelle membrane transport, translation, and mRNA processing. Our results show that model system proteomics experiments are extremely valuable for understanding protein interactions regulating infection in recalcitrant pathogens such as phloem-limited viruses.

Selecting Optimal Peptides for Targeted Proteomic Experiments in Human Plasma Using In Vitro Synthesized Proteins as Analytical Standards.

In targeted proteomics, the development of robust methodologies is dependent upon the selection of a set of optimal peptides for each protein-of-interest. Unfortunately, predicting which peptides and respective product ion transitions provide the greatest signal-to-noise ratio in a particular assay matrix is complicated. Using in vitro synthesized proteins as analytical standards, we report here an empirically driven method for the selection of said peptides in a human plasma assay matrix.

Electron-capture and -transfer dissociation of peptides tagged with tunable fixed-charge groups: structures and dissociation energetics.

Pyridiniummethylcarbonyl moieties that were previously designed on the basis of electronic structure analysis are now utilized as fixed-charge tags with tunable electronic properties to be used for N-terminal peptide derivatization and sequencing by electron-transfer dissociation. Dipeptides AK and KA were derivatized at the peptide N-terminus with 4-dimethylaminopyridinium-N-acetyl (DMAP-ac) and pyridinium-N-acetyl (pyrid-ac) tags of increasing intrinsic recombination energies. Upon the capture of a free electron or electron transfer from fluoranthene anions, (DMAP-ac-AK+H)(2+), (DMAP-ac-KA+H)(2+), (pyrid-ac-AK+H)(2+) and (pyrid-ac-KA+H)(2+) ions, as well as underivatized (AK+2H)(2+), completely dissociated. The fixed-charge tags steered the dissociation upon electron transfer to form abundant backbone N-C(α) bond cleavages, whereas the underivatized peptide mainly underwent H-atom and side-chain losses. Precursor ion structures for the tagged peptides were analyzed by an exhaustive conformational search combined with B3LYP/6-31+G(d,p) geometry optimization and single-point energy calculations in order to select the global energy minima. Structures, relative energies, transition states, ion-molecule complexes, and dissociation products were identified for several charge-reduced species from the tagged peptides. The electronic properties of the charge tags and their interactions with the peptide moieties are discussed. Electrospray ionization and electron-transfer dissociation of larger peptides are illustrated with a DMAP-tagged pentapeptide.

Nectin-like protein 2 defines a subset of T-cell zone dendritic cells and is a ligand for class-I-restricted T-cell-associated molecule.

Dendritic cells (DCs) are a phenotypically and functionally heterogenous population of leukocytes with distinct subsets serving a different set of specialized immune functions. Here we applied an in vitro whole cell panning approach using antibody phage display technology to identify cell-surface epitopes specifically expressed on human blood BDCA3(+) DCs. A single-chain antibody fragment (anti-1F12 scFv) was isolated that recognizes a conserved surface antigen expressed on both human BDCA3(+) DCs and mouse CD8alpha(+) DCs. We demonstrate that anti-1F12 scFv binds Nectin-like protein 2 (Necl2, Tslc1, SynCaM, SgIGSF, or Igsf4), an adhesion molecule involved in tumor suppression, synapse formation, and spermatogenesis. Thus, Necl2 defines a specialized subset of DCs in both mouse and human. We further show that Necl2 binds Class-I-restricted T-cell-associated molecule (CRTAM), a receptor primarily expressed on activated cytotoxic lymphocytes. When present on antigen presenting cells, Necl2 regulates IL-22 expression by activated CD8(+) T-cells. We propose that Necl2/CRTAM molecular pair could regulate a large panel of cell/cell interactions both within and outside of the immune system.

Informatics for protein identification by mass spectrometry.

High throughput protein analysis (i.e., proteomics) first became possible when sensitive peptide mass mapping techniques were developed, thereby allowing for the possibility of identifying and cataloging most 2D gel electrophoresis spots. Shortly thereafter a few groups pioneered the idea of identifying proteins by using peptide tandem mass spectra to search protein sequence databases. Hence, it became possible to identify proteins from very complex mixtures. One drawback to these latter techniques is that it is not entirely straightforward to make matches using tandem mass spectra of peptides that are modified or have sequences that differ slightly from what is present in the sequence database that is being searched. This has been part of the motivation behind automated de novo sequencing programs that attempt to derive a peptide sequence regardless of its presence in a sequence database. The sequence candidates thus generated are then subjected to homology-based database search programs (e.g., BLAST or FASTA). These homology search programs, however, were not developed with mass spectrometry in mind, and it became necessary to make minor modifications such that mass spectrometric ambiguities can be taken into account when comparing query and database sequences. Finally, this review will discuss the important issue of validating protein identifications. All of the search programs will produce a top ranked answer; however, only the credulous are willing to accept them carte blanche.

Studies of ligand-induced site-specific phosphorylation of epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in the regulation of growth in many animal cells, including cancer cells. Phosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR is part of the initial activation process that occurs upon ligand binding, and these phosphotyrosine residues subsequently serve as docking sites for intracellular signaling molecules. To study the phosphorylation on each individual site, EGFR generated from a human epidermoid carcinoma cell line (A431) was analyzed by mass spectrometry. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) was used to identify the tryptic phosphopeptides and their sites of phosphorylation (Y992, Y1045, Y1068, Y1086, S1142, Y1148, and Y1173). Ion intensities for the phosphorylated and unphosphorylated tryptic peptides containing the sites of phosphorylation were measured, and the intensity ratios were used to assess the degree of phosphorylation at each site. Ligand concentrations were varied for epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) as stimuli, and all of the EGFR tyrosine sites were consequently found to exhibit increased levels of phosphorylation, although at different rates and to different extents. Phosphorylation of Y992 appeared to plateau at lower concentrations of ligand, whereas the other sites continued to have increased phosphorylation throughout a wide range of concentrations. Only small differences could be detected between the EGF and the TGF alpha-induced EGFR phosphorylation. Pretreatment of A431 cells with a small molecule EGFR inhibitor nearly eliminated the ligand-induced phosphorylation on all of the sites except for Y992 and Y1068.

Isolation and isotope labeling of cysteine- and methionine-containing tryptic peptides: application to the study of cell surface proteolysis.

Inexpensive methods were developed for isolating and isotopically labeling tryptic peptides that contain either cysteine or methionine. After covalently capturing cysteine-containing peptides with pyridyl disulfide reactive groups on agarose beads, extensive wash steps were applied, and the attached peptides were released using a reducing agent. This approach results in less nonspecifically bound peptides and eliminates the possibility of generating avidin peptide background ions that can arise when using methods based on biotin and avidin (e.g. isotope-coded affinity tag). The thiols were alkylated using either N-ethyl- or N-D5-ethyl-iodoacetamide, both of which can be synthesized in a single step using inexpensive reagents. This isotopic labeling does not greatly increase the peptide mass, nor does it affect the peptide ion charge state in electrospray ionization. In addition, methionine-containing peptides were captured using commercially available methionine-reactive beads, and relative quantitation of peptides was achieved by isotopic labeling of amino groups using activated esters of either nicotinic acid or D4-nicotinic acid. These methods were used to study the metalloprotease-mediated shedding of cell surface proteins from a mouse monocyte cell line that had been treated with a phorbol ester and lipopolysaccharide. In addition to the identification of proteins previously determined to be inducibly shed, three new shed proteins were identified: CD18, ICOS ligand, and tumor endothelial marker 7-related protein.

Searching sequence databases via de novo peptide sequencing by tandem mass spectrometry.

There are many computer programs that can match tandem mass spectra of peptides to database-derived sequences; however, situations can arise where mass spectral data cannot be correlated with any database sequence. In such cases, sequences can be automatically deduced de novo, without recourse to sequence databases, and the resulting peptide sequences can be used to perform homologous nonexact searches of sequence databases. This article describes details on how to implement both a de novo sequencing program called "Lutefisk," and a version of FASTA that has been modified to account for sequence ambiguities inherent in tandem mass spectrometry data.

A proteomic approach for the identification of cell-surface proteins shed by metalloproteases.

Proteolytic cleavage (shedding) of extracellular domains of many membrane proteins by metalloproteases is an important regulatory mechanism used by mammalian cells in response to environmental and physiological changes. Here we describe a proteomic system for analyzing cell surface shedding. The method utilized short-term culture supernatants from induced cells as starting material, followed by lectin-affinity purification, deglycosylation, and polyacrylamide gel electrophoresis separation. Relative quantitation of proteins was achieved via isotope dilution. In this study, a number of proteins already known to be shed were identified from activated monocytes and endothelial cells, thereby validating the method. In addition, a group of proteins were newly identified as being shed. The method provides an unbiased means to screen for shed proteins.