Polymer-based metal nano-coated disposable target for matrix-assisted and matrix-free laser desorption/ionization mass spectrometry.

The ideal MALDI/LDI mass spectrometry sample target for an axial TOF instrument possesses a variety of properties. Primarily, it should be chemically inert to the sample, i.e. analyte, matrix and solvents, highly planar across the whole target, without any previous chemical contact and provide a uniform surface to facilitate reproducible measurements without artifacts from previous sample or matrix compounds. This can be hard to achieve with a metal target, which has to be extensively cleaned every time after use. Any cleaning step may leave residues behind, may change the surface properties due to the type of cleaning method used or even cause microscopic scratches over time hence altering matrix crystallization behavior. Alternatively, use of disposable targets avoids these problems. As each possesses the same surface they therefore have the potential to replace the conventional full metal targets so commonly employed. Furthermore, low cost single-use targets with high planarity promise an easier compliance with GLP guidelines as they alleviate the problem of low reproducibility due to inconsistent sample/matrix crystallization and changes to the target surface properties. In our tests, polymeric metal nano-coated targets were compared to a stainless steel reference. The polymeric metal nano-coated targets exhibited all the performance characteristics for a MALDI MS sample support, and even surpassed the - in our lab commonly used - reference in some aspects like limit of detection. The target exhibits all necessary features such as electrical conductivity, vacuum, laser and solvent compatibility.

Intact cell/intact spore mass spectrometry (IC/ISMS) on polymer-based, nano-coated disposable targets.

Identification and differentiation of microorganisms has and still is a long arduous task, involving culturing of the organism in question on different growth media. This procedure, which is still commonly applied, is an established method, but takes a lot of time, up to several days or even longer. It has thus been a great achievement when other analytical tools like matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were introduced for faster analysis based on the surface protein pattern. Differentiation and identification of human pathogens as well as plant/animal pathogens is of increasing importance in medical care (e.g. infection, sepsis, and antibiotics resistance), biotechnology, food sciences and detection of biological warfare agents. A distinction between microorganisms on the species and strain level was made by comparing peptide/protein profiles to patterns already stored in databases. These profiles and patterns were obtained from the surface of vegetative forms of microorganisms or even their spores by MALDI MS. Thus, an unknown sample can be compared against a database of known pathogens or microorganisms of interest. To benefit from newly available, metal-based disposable microscope-slide format MALDI targets that promise a clean and even surface at a fraction of the cost from full metal targets or MTP (microtiter plate) format targets, IC/ISMS analysis was performed on these and the data evaluated. Various types of bacteria as well as fungal spores were identified unambiguously on this disposable new type of metal nano-coated targets. The method even allowed differentiation between strains of the same species. The results were compared with those gained from using full metal standard targets and found to be equal or even better in several aspects, making the use of disposable MALDI targets a viable option for use in IC/ISMS, especially e.g. for large sample throughput and highly pathogenic species.

Nuclear import of a lipid-modified transcription factor: mobilization of NFAT5 isoform a by osmotic stress.

Lipid-modified transcription factors (TFs) are biomolecular oddities since their reduced mobility and membrane attachment appear to contradict nuclear import required for their gene-regulatory function. NFAT5 isoform a (selected from an in silico screen for predicted lipid-modified TFs) is shown to contribute about half of all endogenous expression of human NFAT5 isoforms in the isotonic state. Wild-type NFAT5a protein is indeed myristoylated and palmitoylated on its transport to the plasmalemma via the endoplasmic reticulum and the Golgi. In contrast, its lipid anchor-deficient mutants as well as isoforms NFAT5b/c are diffusely localized in the cytoplasm without preference to vesicular structures. Quantitative/live microscopy shows the plasmamembrane-bound fraction of NFAT5a moving into the nucleus upon osmotic stress despite the lipid anchoring. The mobilization mechanism is not based on proteolytic processing of the lipid-anchored N-terminus but appears to involve reversible palmitoylation. Thus, NFAT5a is an example of TFs immobilized with lipid anchors at cyotoplasmic membranes in the resting state and that, nevertheless, can translocate into the nucleus upon signal induction.

Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling.

Evolutionary conservation of N-terminal N-myristoylation within protein families indicates significant functional impact of this lipid posttranslational modification for function. In the MYRbase study (Maurer-Stroh et al., (2004) Genome Biology 5, R21), protein families with relevance to asymmetric cell division in animals and the group of plant calcium-dependent protein kinases (CPKs) have surfaced with many predicted myristoylated members. Here, we describe experimental in vitro verification of predicted myristoylation and explore its impact on subcellular localization for these targets in vivo. Our results confirm that, indeed, Numb isoform A, Neuralized isoforms C and D from Drosophila melanogaster and two Neuralized-like homologues from Mus musculus have the capability for N-terminal myristoylation in vitro and in vivo (in fly tissue and in mouse 3T3 cells respectively) whereas other isoforms such as Neuralized A and B have not. The latter two cases are an examples of different potential of various isoforms for posttranslational modifications. Additionally, the Arabidopsis thaliana CDPKs CPK6, CPK9 and CPK13 are shown to be substrates for myristoylation in vitro, which also affects their subcellular localization (in Arabidopsis protoplasts and tobacco leaves). At the same time, CPK6 and CPK13 do not appear to be substrates of a NMT1-like enzyme; the reasons for differing substrate specificities of NMT homologues in plants are derived from the evolutionary divergence of their N-myristoyl transferase sequences. As a methodical advance, we describe a fast and very sensitive technique (compared to traditional autoradiography) for in vitro testing of myristoylation based on thin layer chromatography read-out of the incorporated radioactive myristoyl anchor with subsequent Western blotting detection for protein yield determination using the same membrane.

Towards complete sets of farnesylated and geranylgeranylated proteins.

Three different prenyltransferases attach isoprenyl anchors to C-terminal motifs in substrate proteins. These lipid anchors serve for membrane attachment or protein-protein interactions in many pathways. Although well-tolerated selective prenyltransferase inhibitors are clinically available, their mode of action remains unclear since the known substrate sets of the various prenyltransferases are incomplete. The Prenylation Prediction Suite (PrePS) has been applied for large-scale predictions of prenylated proteins. To prioritize targets for experimental verification, we rank the predictions by their functional importance estimated by evolutionary conservation of the prenylation motifs within protein families. The ranked lists of predictions are accessible as PRENbase (http://mendel.imp.univie.ac.at/sat/PrePS/PRENbase) and can be queried for verification status, type of modifying enzymes (anchor type), and taxonomic distribution. Our results highlight a large group of plant metal-binding chaperones as well as several newly predicted proteins involved in ubiquitin-mediated protein degradation, enriching the known functional repertoire of prenylated proteins. Furthermore, we identify two possibly prenylated proteins in Mimivirus. The section HumanPRENbase provides complete lists of predicted prenylated human proteins-for example, the list of farnesyltransferase targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs) used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domain-containing FLJ32421 (termed BROFTI), and Rab28 (short isoform) as exclusive farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is presented for the selective farnesylation of targets with an evolutionary conserved modification site.

Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo.

BACKGROUND: Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity. RESULTS: We describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase (GST)-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with 3H-labeled anchor precursors. Alternatively, hemagglutinin (HA)-labeled constructs expressed in vivo (in cell culture) can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography (TLC) analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- (or anti-HA-) antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA (variant RhoA63L) including the necessary controls. We show directly that RasD2 is a farnesylation target. CONCLUSION: Savings in time for experimentation and the higher sensitivity for detecting 3H-labeled lipid anchors recommend the TLC-scanning method with purified GST- (or HA-) tagged target proteins as the method of choice for analyzing their prenylation capabilities in vitro and in vivo and, possibly, also for studying the myristoyl and palmitoyl posttranslational modifications.