753 research outputs found

    Determination of pyridoxal-5ā€²-phosphate (PLP)-bonding sites in proteins: a peptide mass fingerprinting approach based on diagnostic tandem mass spectral features of PLP-modified peptides

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    Peptides modified by pyridoxal-5ā€²-phosphate (PLP), linked to a lysine residue via reductive amination, exhibit distinct spectral characteristics in the collision-induced dissociation (CID) tandem mass (MS/MS) spectra that are described here. The MS/MS spectra typically display two dominant peaks whose m/z values correspond to neutral losses of [H 3 PO 4 ] (āˆ’98 Da) and the PLP moiety as [C 8 H 10 NO 5 P] (āˆ’231 Da) from the precursor peptide ion, respectively. Few other peaks are observed. Recognition of this distinct fragmentation behavior is imperative since determining sequences and sites of modifications relies on the formation of amide backbone cleavage products for subsequent interpretation via proteome database searching. Additionally, PLP-modified peptides exhibit suppressed precursor ionization efficiency which diminishes their detection in complex mixtures. Presented here is a protocol which describes an enrichment strategy for PLP-modified peptides combined with neutral loss screening and peptide mass fingerprinting to map the PLP-bonding site in a known PLP-dependent protein. This approach represents an efficient alternative to site-directed mutagenesis which has been the traditional method used for PLP-bonding site localization in proteins. Copyright Ā© 2009 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/64342/1/4270_ftp.pd

    Wnt5a induces ROR1 to complex with HS1 to enhance migration of chronic lymphocytic leukemia cells.

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    ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1Pā†’ļø€A mutants, ROR1P(841)A had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration

    Barrel swirl breakdown in spark-ignition engines: Insights from particle image velocimetry measurements

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    This is an article from the journal, Proceedings of the Institution of Mechanical Engineers, Part D: Journal of Automobile Engineering [Ā© IMechE ]. It is also available at: http://dx.doi.org/10.1243/0954407991527134Particle image velocimetry (PIV) has been used here to study the formation and breakdown of barrel swirl ('tumble') in a production geometry, four-stroke, four-valve, motored, spark-ignition, optically accessed internal combustion (IC) engine. The barrel swirl ratio (BSR) of the cylinder head could be enhanced by means of a port face inducer gasket so that the flow processes taking place at low and high swirl ratios could be investigated conveniently. Double-exposed images from planes both parallel and perpendicular to the cylinder axis were recorded at selected crank angles through the induction and compression strokes at a motored engine speed of 1000 r/min, with a wide open throttle, for both high and low BSR cases. The recorded images were interrogated by digital autocorrelation to give two-dimensional maps of instantaneous velocity. In both high and low BSR cases, a barrel or tumbling vortex motion is generated during induction, which is shown to persist throughout the majority of the compression stroke. The details of barrel swirl formation during induction and its subsequent modification during compression, however, differ strongly between the two cases. These differences can be explained qualitatively in terms of two main events; the first being competition during induction between vortices of unequal strength and the second being competition between the large-scale swirl motion and the local flow field generated by piston motion during compression. In the low barrel swirl case, significant dissipation occurs owing to these interactions and consequently the large-scale motion exhibits lower mean velocities and undergoes significant distortion. In the case of high BSR, the competition effects are minimized and a single ordered vertical vortex exhibiting high velocity magnitudes is observed to avoid piston induced distortion. It then moves into the apex of the pent roof combustion chamber where it survives as a single ordered vortex until at least 40Ā° crank angle (CA) before top dead centre (TDC). Limitations and developments of the PIV technique are discussed

    Hydrazines as versatile chemical biology probes and drug-discovery tools for cofactor-dependent enzymes [preprint]

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    Known chemoproteomic probes generally use warheads that tag a single type of amino acid or modified form thereof to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electron-poor enzyme cofactors, transient intermediates and chemically-labile regulatory modifications, but probes for such species are underdeveloped. Here, we have innovated a versatile class of chemoproteomic probes for this less charted hemisphere of the proteome by using hydrazine as the common chemical warhead. Its electron-rich nature allows it to react by both polar and radicaloid mechanisms and to target multiple, pharmacologically important functional classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment can be blocked by active-site-directed inhibitors, and elaboration of the warhead supports connection of a target to a lead compound. The capacity of substituted hydrazines to profile, discover and inhibit diverse cofactor-dependent enzymes enables cell and tissue imaging and makes this platform useful for enzyme and drug discovery

    Computational refinement of post-translational modifications predicted from tandem mass spectrometry

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    Motivation: A post-translational modification (PTM) is a chemical modification of a protein that occurs naturally. Many of these modifications, such as phosphorylation, are known to play pivotal roles in the regulation of protein function. Henceforth, PTM perturbations have been linked to diverse diseases like Parkinson's, Alzheimer's, diabetes and cancer. To discover PTMs on a genome-wide scale, there is a recent surge of interest in analyzing tandem mass spectrometry data, and several unrestrictive (so-called ā€˜blindā€™) PTM search methods have been reported. However, these approaches are subject to noise in mass measurements and in the predicted modification site (amino acid position) within peptides, which can result in false PTM assignments
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