EMSL: Ljiljana Pasa Tolic's Publications

Ream TS, JR Haag, AT Wierzbicki, CD Nicora, AD Norbeck, JK Zhu, G Hagen, TJ Guilfoyle, L Pasa-Tolic, and CS Pikaard. 2009. "Subunit Compositions of the RNA-Silencing Enzymes Pol IV and Pol V Reveal Their Origins as Specialized Forms of RNA Polymerase II ." Molecular Cell 33(2):192-203. doi:doi:10.1016/j.molcel.2008.12.015 Abstract In addition to RNA polymerases I, II and III, which are multi-subunit RNA polymerases found in all eukaryotes, plants have catalytic subunits for two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V (formerly Pol IVa and Pol IVb, respectively). Pol IV and Pol V play non-redundant roles in siRNA-directed DNA methylation and gene silencing pathways.

Springer DL, JH Miller, SL Spinelli, L Pasa-Tolic, SO Purvine, DS Daly, RC Zangar, S Jin, N Blumberg, CW Francis, MB Taubman, AE Casey, SD Wittlin, and RP Phipps. 2009. "Platelet Proteome Changes Associated with Diabetes and During Platelet Storage for Transfusion." Journal of Proteome Research 8(5):2261-2272. Abstract Human platelets play a key role in homeostasis and thrombosis and have recently emerged as key regulators of inflammation. Platelets stored for transfusion produce pro-thrombotic and pro-inflammatory mediators implicated in adverse transfusion reactions. Correspondingly, these mediators are central players in pathological conditions including cardiovascular disease, the major cause of death in diabetics. In view of this, a mass spectrometry based proteomics study was performed on platelets collected from healthy and type-2 diabetics stored for transfusion. Strikingly, our innovative and sensitive proteomic approach identified 146 proteins that were either up- or down-regulated in type-2 diabetics relative to non-diabetic controls, 151 proteins whose abundances changed during a 5-day storage period and 22 proteins whose abundance changed after 5-days of storage were only observed in samples from diabetics. Notably our studies are the first to characterize the proteome of platelets from diabetics before and after storage for transfusion. These identified differences allow us to formulate new hypotheses and experimentation to improve clinical outcomes by targeting "high risk platelets" that render platelet transfusion less effective or even unsafe.

Tolmachev AV, EW Robinson, S Wu, L Pasa-Tolic, and RD Smith. 2009. "FT-ICR MS optimization for the analysis of intact proteins." International Journal of Mass Spectrometry 287(1-3 SP ISS):32-38. doi:10.1016/j.ijms.2008.10.010 Abstract Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) remains the technique of choice for the analysis of intact proteins from complex biological systems, i.e. top-down proteomics. Recently, we have implemented a compensated open cylindrical ion trapping cell into a 12 T FT-ICR mass spectrometer. This new cell has previously demonstrated improved sensitivity, dynamic range, and mass measurement accuracy for the analysis of relatively small tryptic peptides. These improvements are due to the improved trapping potential of the cell which is significantly closer to the ideal harmonic trapping potential. Here we report the instrument optimization for the analysis of large macro-molecular ions, such as proteins. Also, presented are first principle theoretical considerations to account for different optimum conditions for the analysis of large macro-molecules. The proposed high energy ion loss mechanism is further supported by experimental results of bovine ubiquitin and serum albumin. We find that the analysis of large macro-molecules can be significantly improved by the further reduction of pressure in the ion trapping cell. This will reduce the impact of the high energy ion loss mechanism and enable increased sensitivity and mass measurement accuracy to be realized without compromising resolution. Further, these results appear to be applicable to FTMS in general, and the high energy ion loss mechanism applies to Orbitrap mass analyzers as well.

Umar AN, H Kang, AM Timmermans, MP Look, ME Meijer-van Gelder, MA den Bakker, N Jaitly, JW Martens, TM Luider, JA Foekens, and L Pasa-Tolic. 2009. "Identification of a putative protein profile associating with tamoxifen therapy resistance in breast cancer." Molecular & Cellular Proteomics. MCP 8(6):1278-1294. doi:10.1074/mcp.M800493-MCP200 Abstract Tamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy-resistance in breast cancer, using nanoLC coupled with FTICR MS. Comparative proteome analysis was performed on ~5,500 pooled tumor cells (corresponding to ~550 ng protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n=24 and n=27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag (AMT) reference databases.

Wu S, NM Lourette, N Tolic, R Zhao, R Robinson, AV Tolmachev, RD Smith, and L Pasa-Tolic. 2009. "An integrated top-down and bottom-up strategy for broadly characterizing protein isoforms and modifications." Journal of Proteome Research 8(3):1347-1357. Abstract We present an integrated top-down and bottom-up approach facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs high resolution reversed phase (RP) LC separations coupled on-line with a 12T Fourier transform ion cyclotron resonance (FTICR) spectrometer to profile and tentatively identify modified proteins, using detected intact protein masses in conjunction with bare protein identifications from the bottom-up analysis of the same fraction. Selected identifications are incorporated into a target ion list for subsequent offline gas phase fragmentation that uses only an aliquot of the original fraction used for bottom-up analysis.

Wu S, F Yang, R Zhao, N Tolic, EW Robinson, DG Camp, II, RD Smith, and L Pasa-Tolic. 2009. "An integrated workflow for characterizing intact phosphoproteins from complex mixtures." Analytical Chemistry 81(11):4210-4219. doi:10.1021/ac802487q Abstract The phosphorylation of any site on a given protein can affect its activity, degradation rate, ability to dock with other proteins or bind divalent cations, and/or its localization. These effects can operate within the same protein; in fact, multisite phosphorylation is a key mechanism for achieving signal integration in cells. Hence, knowing the overall phosphorylation signature of a protein is essential for understanding the "state" of a cell. However, current technologies to monitor the phosphorylation status of proteins are inefficient at determining the relative stoichiometries of phosphorylation at multiple sites. Here we report a new capability for comprehensive liquid chromatography-mass spectrometry (LC-MS) analysis of intact phosphoproteins. The technology platform built upon integrated bottom-up and top-down approach that is facilitated by intact protein reversed-phase (RP)LC concurrently coupled with Fourier transform ion cyclotron resonance (FTICR) MS and fraction collection.

Yang F, S Wu, DL Stenoien, R Zhao, ME Monroe, MA Gritsenko, SO Purvine, AD Polpitiya, N Tolic, Q Zhang, AD Norbeck, DJ Orton, RJ Moore, K Tang, GA Anderson, L Pasa-Tolic, DG Camp, II, and RD Smith. 2009. "Combined Pulsed-Q dissociation and electron transfer dissociation for identification and quantitation of iTRAQ–labeled phosphopeptides." Analytical Chemistry 81(10):4137-4143. doi:10.1021/ac802605m Abstract Multiplex isobaric tags for relative and absolute quantification (iTRAQ) enable high-throughput quantification of peptides via reporter ion signals in the low mass range of tandem mass spectra. A challenging but highly promising application is to analyze iTRAQ-labeled peptides using a sensitive linear ion trap mass spectrometer (LTQ-MS) and pulsed Q dissociation (PQD), a form of ion trap collision activated dissociation (CAD) designed to allow detection of low mass-to-charge fragment ions. Electron dissociation transfer (ETD), on the other hand, is complementary to PQD and is especially useful for sequencing peptides containing post-translational modifications (PTMs). Here, we developed an integrated workflow for robust and accurate quantitative identification of iTRAQ labeled phosphopeptides that integrates the PQD and ETD fragmentation methods together with PQD optimization, data management and bioinformatics tools. Analysis of the phosphoproteome of human fibroblast cells demonstrated that this hybrid mode is superior to either PQD or ETD alone for phosphopeptide identification and quantitation. The combined PQD/ETD approach can qualitatively identify additional phosphopeptides than ETD alone and PQD information can provide better quantitation of ETD identified iTRAQ-labeled phosphopeptides.

Brewer HM, AD Norbeck, JN Adkins, NP Manes, C Ansong, L Shi, Y Rikihisa, T Kikuchi, S Wong, RD Estep, F Heffron, L Pasa-Tolic, and RD Smith. 2008. "Optimization of proteomic sample preparation procedures for comprehensive protein characterization of pathogenic systems." Journal of Biomolecular Techniques:JBT 19(5):285-295. Abstract The elucidation of critical functional pathways employed by pathogens and hosts during an infectious cycle is both challenging and central to our understanding of infectious diseases. In recent years, mass spectrometry-based proteomics has been used as a powerful tool to identify key pathogenesis-related proteins and pathways. Despite the analytical power of mass spectrometry-based technologies, samples must be appropriately prepared to characterize the functions of interest (e.g. host-response to a pathogen or a pathogen-response to a host). The preparation of these protein samples requires multiple decisions about what aspect of infection is being studied, and it may require the isolation of either host and/or pathogen cellular material.

Shen Y, N Tolic, KK Hixson, SO Purvine, L Pasa-Tolic, W Qian, JN Adkins, RJ Moore, and RD Smith. 2008. "Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags." Analytical Chemistry 80(6):1871-82. doi:10.1021/ac702328x Abstract Identifying proteins correctly and with known levels of confidence remain as significant challenges for proteomics. Random or decoy peptide databases are increasingly being used to estimate the false discovery rate (FDR), e.g., from liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of tryptic digests. We show that this approach can significantly underestimate the FDR, and describe an approach for more confident protein identifications that uses unique partial sequences derived from a combination of database searching and de novo-style data analyses of high precision MS/MS data. Applied to a Saccharomyces cerevisiae tryptic digest, the approach provided 3,132 confident peptide identifications (~5% modified in some fashion), covering 575 proteins with an estimated zero FDR. The conventional approach provided 3,359 peptide identifications and 656 proteins with 0.3% FDR based upon a decoy database analysis. However, the present approach revealed ~5% of the 3,359 identifications to be incorrect, and many more as potentially ambiguous, (e.g., due to not considering certain amino acid substitutions and modifications). In addition, 677 peptides and 39 proteins were identified that had been missed by conventional analysis, including non-tryptic peptides, peptides with various expected/unexpected chemical modifications, known/unknown posttranslational modifications, single nucleotide polymorphisms or gene encoding errors, and multiple modifications of individual peptides.

Tolmachev AV, EW Robinson, S Wu, H Kang, NM Lourette, L Pasa-Tolic, and RD Smith. 2008. " Trapped-ion cell with improved DC potential harmonicity for FT-ICR MS." Journal of the American Society for Mass Spectrometry 19(4):586 - 597. doi:10.1016/j.jasms.2008.01.006 Abstract The trapped-ion cell is a key component critical for optimal performance in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). We have upgraded our 12 Tesla FT-ICR instrument with a new open cylindrical cell that includes four additional cylindrical segments that serve as compensation electrodes. The DC potential on the additional segments can be set to specific pre-calculated values to suppress DC trapping field anharmonicity, in an effort to improve coherence of the ion cyclotron motion and minimize deviations from the calibration function of the ideal cell. Alternatively, the compensation potentials can be set equal to potentials of adjacent cell electrodes, which creates a DC potential distribution equivalent to that of a regular open cylindrical cell. The initial experimental characterization of both the compensated and open cell configurations was performed using ESI direct infusion of a peptide mixture. Operating the compensated cell at increased post-excitation radii resulted in improved mass measurement accuracy together with increased signal intensity, while the regular configuration exhibited peak splitting and reduced signal life time under these operating conditions. The observed improvement of the compensated cell performance was consistent with the expected behavior due to the improved DC potential harmonicity. These results confirm that the trapping DC potential harmonicity is significant for optimizing FT-ICR MS performance.