@article{galvao_kota_soderblom_goshe_boss_2008, title={Characterization of a new family of protein kinases from Arabidopsis containing phosphoinositide 3/4-kinase and ubiquitin-like domains}, volume={409}, ISSN={["1470-8728"]}, DOI={10.1042/bj20070959}, abstractNote={At least two of the genes predicted to encode type II PI4K (phosphoinositide 4-kinase) in Arabidopsis thaliana (thale cress), namely AtPI4Kγ4 and AtPI4Kγ7, encode enzymes with catalytic properties similar to those of members of the PIKK (phosphoinositide kinase-related kinase) family. AtPI4Kγ4 and AtPI4Kγ7 undergo autophosphorylation and phosphorylate serine/threonine residues of protein substrates, but have no detectable lipid kinase activity. AtPI4Kγ4 and AtPI4Kγ7 are members of a subset of five putative AtPI4Ks that contain N-terminal UBL (ubiquitin-like) domains. In vitro analysis of AtPI4Kγ4 indicates that it interacts directly with, and phosphorylates, two proteins involved in the ubiquitin–proteasome system, namely UFD1 (ubiquitin fusion degradation 1) and RPN10 (regulatory particle non-ATPase 10). On the basis of the present results, we propose that AtPI4Kγ4 and AtPI4Kγ7 should be designated UbDKγ4 and UbDKγ7 (ubiquitin-like domain kinases γ4 and γ7). These UBL-domain-containing AtPI4Ks correspond to a new PIKK subfamily of protein kinases. Furthermore, UFD1 and RPN10 phosphorylation represents an additional mechanism by which their function can be regulated.}, journal={BIOCHEMICAL JOURNAL}, author={Galvao, Rafaelo M. and Kota, Uma and Soderblom, Erik J. and Goshe, Michael B. and Boss, Wendy F.}, year={2008}, month={Jan}, pages={117–127} } @article{whitehurst_soderblom_west_hernandez_goshe_brown_2007, title={Location and role of free cysteinyl residues in the Sindbis virus E1 and E2 glycoproteins}, volume={81}, ISSN={["0022-538X"]}, DOI={10.1128/JVI.02859-06}, abstractNote={ABSTRACT Sindbis virus is a single-stranded positive-sense RNA virus. It is composed of 240 copies of three structural proteins: E1, E2, and capsid. These proteins form a mature virus particle composed of two nested T=4 icosahedral shells. A complex network of disulfide bonds in the E1 and E2 glycoproteins is developed through a series of structural intermediates as virus maturation occurs (M. Mulvey and D. T. Brown, J. Virol. 68:805-812, 1994; M. Carleton et al., J. Virol. 71:1558-1566, 1997). To better understand the nature of this disulfide network, E1 and E2 cysteinyl residues were labeled with iodoacetamide in the native virus particle and analyzed by liquid chromatography-tandem mass spectrometry. This analysis identified cysteinyl residues of E1 and E2, which were found to be label accessible in the native virus particle, as well as those that were either label inaccessible or blocked by their involvement in disulfide bonds. Native virus particles alkylated with iodoacetamide demonstrated a 4-log decrease in viral infectivity. This suggests that the modification of free cysteinyl residues results in the loss of infectivity by destabilizing the virus particle or that a rearrangement of disulfide bonds, which is required for infectivity, is blocked by the modification. Although modification of these residues prevented infectivity, it did not alter the ability of virus to fuse cells after exposure to acidic pH; thus, modification of free cysteinyl residues biochemically separated the process of infection from the process of membrane fusion.}, number={12}, journal={JOURNAL OF VIROLOGY}, author={Whitehurst, Christopher B. and Soderblom, Erik J. and West, Michelle L. and Hernandez, Raquel and Goshe, Michael B. and Brown, Dennis T.}, year={2007}, month={Jun}, pages={6231–6240} } @article{soderblom_bobay_cavanagh_goshe_2007, title={Tandem mass spectrometry acquisition approaches to enhance identification of protein-protein interactions using low-energy collision-induced dissociative chemical crosslinking reagents}, volume={21}, ISSN={["0951-4198"]}, DOI={10.1002/rcm.3213}, abstractNote={AbstractChemical crosslinking combined with mass spectrometry is a useful tool for studying the topological organization of multiprotein interactions, but it is technically challenging to identify peptides involved in a crosslink using tandem mass spectrometry (MS/MS) due to the presence of product ions originating from both peptides within the same crosslink. We have previously developed a novel set of collision‐induced dissociative chemical crosslinking reagents (CID‐CXL reagents) that incorporate a labile bond within the linker which readily dissociates at a single site under low‐energy collision‐induced dissociation (CID) to enable independent isolation and sequencing of the crosslinked peptides by traditional MS/MS and database searching. Alternative low‐energy CID events were developed within the in‐source region by increasing the multipole DC offset voltage (ISCID) or within the ion trap by increasing the collisional excitation (ITCID). Both dissociation events, each having their unique advantages, occur without significant backbone fragmentation to the peptides, thus permitting subsequent CID to be applied to these distinct peptide ions for generation of suitable product ion spectra for database searching. Each approach was developed and applied to a chemical crosslinking study involving the N‐terminal DNA‐binding domain of AbrB (AbrBN), a transition‐state regulator in Bacillus subtilis. A total of thirteen unique crosslinks were identified using the ITCID approach which represented a significant improvement over the eight unique crosslinks identified using the ISCID approach. The ability to segregate intrapeptide and interpeptide crosslinks using ITCID represents the first step towards high‐throughput analysis of protein‐protein crosslinks using our CID‐CXL reagents. Copyright © 2007 John Wiley & Sons, Ltd.}, number={21}, journal={RAPID COMMUNICATIONS IN MASS SPECTROMETRY}, author={Soderblom, Erik J. and Bobay, Benjamin G. and Cavanagh, John and Goshe, Michael B.}, year={2007}, pages={3395–3408} } @article{liu_soderblom_goshe_2006, title={A mass spectrometry-based proteomic approach to study Marek's Disease Virus gene expression}, volume={135}, ISSN={0166-0934}, url={http://dx.doi.org/10.1016/j.jviromet.2006.02.001}, DOI={10.1016/j.jviromet.2006.02.001}, abstractNote={Marek's Disease Virus (MDV) is an avian herpesvirus that causes a lymphoproliferative disorder in chickens. MDV transitions between a lytic phase in which new viruses are produced and a latent phase in which the virus lays dormant. The mechanism controlling this lytic-to-latent switch remains unclear. To better understand the lytic phase of MDV infection, a mass spectrometry-based strategy was developed to identify viral proteins and to qualitatively examine their abundance in lytically infected chicken embryo fibroblast (CEF) cells. A combination of strong cation exchange chromatography (SCXC) and microcapillary reversed-phase liquid chromatography-tandem mass spectrometry (murpLC/MS/MS) was used to resolve peptides from tryptic digests of MDV-infected CEF cell lysates. Peptides were identified by searching the tandem mass spectra against a protein database containing both MDV proteins and all currently available Gallus gallus proteins using the SEQUEST algorithm. A total of 427 MDV peptides, corresponding to 82 unique proteins, were identified, with 56 of them detected with at least two unique peptides. Overall, nearly 80% of all putative MDV proteins expressed in infected CEF cells were identified. We anticipate that this approach will be a viable method for determining how viral and host proteome changes occurring in Marek's Disease pathogenesis regulate the switch between the lytic and latent phases of the MDV life cycle.}, number={1}, journal={Journal of Virological Methods}, publisher={Elsevier BV}, author={Liu, Hsiao-Ching S. and Soderblom, Erik J. and Goshe, Michael B.}, year={2006}, month={Jul}, pages={66–75} } @article{soderblom_goshe_2006, title={Collision-induced dissociative chemical cross-linking reagents and methodology: Applications to protein structural characterization using tandem mass spectrometry analysis}, volume={78}, ISSN={["0003-2700"]}, DOI={10.1021/ac0613840}, abstractNote={Chemical cross-linking combined with mass spectrometry is a viable approach to study the low-resolution structure of protein and protein complexes. However, unambiguous identification of the residues involved in a cross-link remains analytically challenging. To enable a more effective analysis across various MS platforms, we have developed a novel set of collision-induced dissociative cross-linking reagents and methodology for chemical cross-linking experiments using tandem mass spectrometry (CID-CXL-MS/MS). These reagents incorporate a single gas-phase cleavable bond within their linker region that can be selectively fragmented within the in-source region of the mass spectrometer, enabling independent MS/MS analysis for each peptide. Initial design concepts were characterized using a synthesized cross-linked peptide complex. Following verification and subsequent optimization of cross-linked peptide complex dissociation, our reagents were applied to homodimeric glutathione S-transferase and monomeric bovine serum albumin. Cross-linked residues identified by our CID-CXL-MS/MS method were in agreement with published crystal structures and previous cross-linking studies using conventional approaches. Common LC/MS/MS acquisition approaches such as data-dependent acquisition experiments using ion trap mass spectrometers and product ion spectral analysis using SEQUEST were shown to be compatible with our CID-CXL-MS/MS reagents, obviating the requirement for high resolution and high mass accuracy measurements to identify both intra- and interpeptide cross-links.}, number={23}, journal={ANALYTICAL CHEMISTRY}, author={Soderblom, Erik J. and Goshe, Michael B.}, year={2006}, month={Dec}, pages={8059–8068} } @article{wang_goshe_soderblom_phinney_kuchar_li_asami_yoshida_huber_clouse_2005, title={Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase}, volume={17}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.105.031393}, abstractNote={Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography–tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.}, number={6}, journal={PLANT CELL}, author={Wang, XF and Goshe, MB and Soderblom, EJ and Phinney, BS and Kuchar, JA and Li, J and Asami, T and Yoshida, S and Huber, SC and Clouse, SD}, year={2005}, month={Jun}, pages={1685–1703} } @article{feeney_soderblom_goshe_clark_2006, title={Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker}, volume={47}, ISSN={1046-5928}, url={http://dx.doi.org/10.1016/j.pep.2005.10.005}, DOI={10.1016/j.pep.2005.10.005}, abstractNote={Coupled with over-expression in host organisms, fusion protein systems afford economical methods to obtain large quantities of target proteins in a fast and efficient manner. Some proteases used for these purposes cleave C-terminal to their recognition sequences and do not leave extra amino acids on the target. However, they are often inefficient and are frequently promiscuous, resulting in non-specific cleavages of the target protein. To address these issues, we created a fusion protein system that utilizes a highly efficient enzyme and leaves no residual amino acids on the target protein after removal of the affinity tag. We designed a glutathione S-transferase (GST)-fusion protein vector with a caspase-3 consensus cleavage sequence located between the N-terminal GST tag and a target protein. We show that the enzyme efficiently cleaves the fusion protein without leaving excess amino acids on the target protein. In addition, we used an engineered caspase-3 enzyme that is highly stable, has increased activity relative to the wild-type enzyme, and contains a poly-histidine tag that allows for efficient removal of the enzyme after cleavage of the fusion protein. Although we have developed this system using a GST tag, the system is amenable to any commercially available affinity tag.}, number={1}, journal={Protein Expression and Purification}, publisher={Elsevier BV}, author={Feeney, Brett and Soderblom, Erik J. and Goshe, Michael B. and Clark, A. Clay}, year={2006}, month={May}, pages={311–318} }