EMSL: Ron Moore's Publications

Marginean I, RT Kelly, RJ Moore, DC Prior, BL Lamarche, K Tang, and RD Smith. 2009. "Selection of the Optimum Electrospray Voltage for Gradient Elution LC-MS Measurements." Journal of the American Society for Mass Spectrometry 20(4):682-688. Abstract Changes in liquid composition during gradient elution liquid chromatography (LC) and mass spectrometry (MS) analyses affect the electrospray operation. To establish methodologies for judicious selection of the electrospray voltage, we monitored in real-time the effect of the LC gradient on the spray current. The optimum range of the electrospray voltage shifted to lower values as the concentration of organic solvent in the eluent increased during reversed-phase LC analyses. These results provided the means to rationally select the voltage that ensured successful electrospray operation throughout gradient elution LC-MS experiments. A small run-to-run drift in the spray current was observed for electrosprays operated at constant voltage. This could be the result of fouling or degradation of the electrospray emitter, which affected the electric field driving the electrospray. Algorithms using feedback from spray current measurements to maintain the electrospray voltage within the optimum operating range throughout gradient elution LC-MS were evaluated. The electrospray operation with voltage regulation and at constant, judiciously selected voltage during gradient elution LC-MS measurements produced data with similar reproducibility.

Qian W, T Liu, VA Petyuk, MA Gritsenko, BO Petritis, AD Polpitiya, A Kaushal, W Xiao, CC Finnerty, MG Jescheke, N Jaitly, ME Monroe, RJ Moore, LL Moldawer, RW Davis, RG Tompkins, DN Hemdon, DG Camp, II, and RD Smith. 2009. "Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an O-18-Labeled “Universal” Reference Sample ." Journal of Proteome Research 8(1):290-299. Abstract Quantitative comparison of protein abundances across a relatively large number of patient samples is an important challenge for clinical proteomic applications. Herein we describe a dual-quantitation strategy that allows the simultaneous integration of complementary label-free and stable isotope labeling based approaches without increasing the number of LC-MS analyses. The approach utilizes a stable isotope 18O-labeled “universal” reference sample as a comprehensive set of internal standards spiked into each individually processed unlabeled patient sample. The quantitative data are based on both the direct 16O-MS intensities for label-free quantitation and the 16O/18O isotopic peptide pair ratios that compare each patient sample to the identical labeled reference. The effectiveness of this dual-quantitation approach for large scale quantitative proteomics is demonstrated by the application to a set of 38 clinical plasma samples from surviving and non-surviving severe burn patients. With the coupling of immunoaffinity depletion, cysteinyl-peptide enrichment based fractionation, high resolution LC-MS measurements, and the dual-quantitation approach, a total of 318 proteins were confidently quantified with at least two peptides and 263 proteins were quantified by both approaches. The strategy also enabled a direct comparison between the two approaches with the labeling approach showing significantly better precision in quantitation while the label-free approach resulted in more protein identifications. The relative abundance differences determined by the two approaches also show strong correlation. Finally, the dual-quantitation strategy allowed us to identify more candidate protein biomarkers, illustrating the complementary nature of the two quantitative methods.

Weber TJ, LK Opresko, DM Waisman, GJ Newton, RD Quesenberry, N Bollinger, RJ Moore, and RD Smith. 2009. "Regulation of the Low Dose Radiation Paracrine-Specific Anchorage-Independent Growth Response by Annexin A2." Radiation Research 172(1):96-105. doi:10.1667/RR1220.1 Abstract ABSTRACT-Here we identify release of annexin A2 into the culture medium in response to low dose X-ray radiation exposure and establish functional linkages to an established paracrine factor-mediated anchorage-independent growth response. Using a standard bicameral coculture model, we observe that annexin A2 levels associated with non-irradiated neighboring cells seeded in the lower chamber (annexin A2 silenced [shRNA] JB6 cells) are increased upon coculture with irradiated (10-50 cGy) JB6 cells seeded in the upper chamber, relative to coculture with sham exposed JB6 cells seeded in the upper chamber, suggesting that annexin A2 released into the medium is capable of communicating in a paracrine fashion. Using a previously established coculture model, we observed that the paracrine factor-mediated anchorage-independent growth response to low dose X-ray radiation is markedly reduced when irradiated annexin A2 silenced (shRNA) JB6 cells are used, relative to coculture with irradiated annexin A2 competent vector control counterparts. These observations suggest that annexin A2 is functionally linked to the radiation paracrine factor-specific anchorage-independent growth response in JB6 cells.

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.

Ding J, CM Sorensen, N Jaitly, H Jiang, DJ Orton, ME Monroe, RJ Moore, RD Smith, and TO Metz. 2008. "Application of the accurate mass and time tag approach in studies of the human blood lipidome." Journal of Chromatography B 871(2):243-252. doi:10.1016/j.jchromb.2008.04.040 Abstract We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput quantitative LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance. In addition, we also report on the optimization of a reversed-phase LC method for the separation of lipids in these sample types.

Livesay EA, K Tang, BK Taylor, MA Buschbach, DF Hopkins, BL Lamarche, R Zhao, Y Shen, DJ Orton, RJ Moore, RT Kelly, HR Udseth, and RD Smith. 2008. "Fully automated four-column capillary LC-MS system for maximizing throughput in proteomic analyses." Analytical Chemistry 80(1):294-302. doi:10.1021/ac701727r Abstract We describe a 4-column, high-pressure capillary liquid chromatography (LC) system for robust, high-throughput LC-MS(/MS) analyses. This system performs multiple LC separations in parallel, but staggers each of them such that the data-rich region of each separation is sampled sequentially. By allowing nearly continuous data acquisition, this design maximizes the use of the mass spectrometer. Each analytical column is connected to a corresponding ESI emitter in order to avoid the use of post-column switching and associated dead volume issues. Encoding translation stages are employed to sequentially position the emitters at the MS inlet. The high reproducibility of this system is demonstrated using consecutive analyses of global tryptic digest of the microbe Shewanella oneidensis.

Lopez-Ferrer D, K Petritis, KK Hixson, TH Heibeck, RJ Moore, ME Belov, DG Camp, II, and RD Smith. 2008. "Application of pressurized solvents for ultrafast trypsin hydrolysis in proteomics: Proteomics on the fly." Journal of Proteome Research 7(8):3276-3281. doi:10.1021/pr7008077 Abstract A new method for rapid proteolytic digestion of proteins under high pressure that uses pressure cycling technology in the range of 5 to 35 kpsi was demonstrated for proteomic analysis. Successful in-solution digestions of single proteins and complex protein mixtures were achieved in 60 s and then analyzed by reversed phase liquid chromatography-electrospray ionization ion trap-mass spectrometry. Method performance in terms of the number of Shewanella oneidensis peptides and proteins identified in a shotgun approach was evaluated relative to a traditional “overnight” sample preparation method. Advantages of the new method include greatly simplified sample processing, easy implementation, no cross contamination among samples, and cost effectiveness.

Lopez-Ferrer D, TH Heibeck, K Petritis, KK Hixson, W Qian, ME Monroe, AM Mayampurath, RJ Moore, ME Belov, DG Camp, II, and RD Smith. 2008. "Rapid Sample Processing For LC-MS Based Quantitative Proteomics Using High Intensity Focused Ultrasounds." Journal of Proteome Research 7(9):3860-3867. doi:10.1021/pr800161x Abstract A new sample processing workflow that uses high intensity focused ultrasound to rapidly reduce and alkylate cysteines, digest proteins and then label peptides with 18O was developed for quantitative proteomics applications. Each step was individually refined to minimize reaction times, peptide loses and undesired by-products or modifications. By using this novel workflow, mouse plasma proteins were successfully denatured, alkylated, in-solution digested, and 18O labelled in < 10 min for subsequent analysis by liquid chromatography-electrospray ionization high resolution mass spectrometry. Performance was evaluated in terms of the number of mouse plasma peptides and proteins identified in a shotgun approach and the quantitative dynamic range. The results were compared with previously published results obtained using conventional sample preparation methods and were found to be similar. Advantages of the new method include greatly simplified and accelerated sample processing, as well as being readily amenable to automation.

Manes NP, RD Estep, HM Mottaz, RJ Moore, TRW Clauss, ME Monroe, X Du, JN Adkins, S Wong, and RD Smith. 2008. "Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions." Journal of Proteome Research 3:960-8. doi:10.1021/pr070432+ Abstract Orthopoxviruses are the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, virulent (monkeypox virus) and benign (vaccinia virus) orthopoxviruses were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by reversed-phase LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® and X! Tandem resulted in the identification of hundreds of monkeypox, vaccinia, and copurified host proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially expressed orthopoxvirus genes are discussed.

Metz TO, W Qian, JM Jacobs, MA Gritsenko, RJ Moore, AD Polpitiya, ME Monroe, DG Camp, II, PW mueller, and RD Smith. 2008. "Application of proteomics in the discovery of candidate protein biomarkers in a Diabetes Autoantibody Standardization Program sample subset ." Journal of Proteome Research 7(2):698-707. doi:10.1021/pr700606w Abstract Objective. Before biomarkers predictive of type 1 diabetes can be evaluated in proficiency evaluations, they must be identified and validated in initial, exploratory studies. Hypothesis-driven comparative studies may be performed to identify candidate biomarkers but are limited to the current knowledge of metabolic, signaling, and inflammatory pathways in the context of type 1 diabetes. Alternatively, untargeted “-omics” approaches may be employed in profiling studies to identify candidate biomarkers of type 1 diabetes.