@article{andrews_dean_hawkridge_muddiman_2011, title={Improving Proteome Coverage on a LTQ-Orbitrap Using Design of Experiments}, volume={22}, ISSN={["1879-1123"]}, DOI={10.1007/s13361-011-0075-2}, abstractNote={Design of experiments (DOE) was used to determine improved settings for a LTQ-Orbitrap XL to maximize proteome coverage of Saccharomyces cerevisiae. A total of nine instrument parameters were evaluated with the best values affording an increase of approximately 60% in proteome coverage. Utilizing JMP software, 2 DOE screening design tables were generated and used to specify parameter values for instrument methods. DOE 1, a fractional factorial design, required 32 methods fully resolving the investigation of six instrument parameters involving only half the time necessary for a full factorial design of the same resolution. It was advantageous to complete a full factorial design for the analysis of three additional instrument parameters. Measured with a maximum of 1% false discovery rate, protein groups, unique peptides, and spectral counts gauged instrument performance. Randomized triplicate nanoLC-LTQ-Orbitrap XL MS/MS analysis of the S. cerevisiae digest demonstrated that the following five parameters significantly influenced proteome coverage of the sample: (1) maximum ion trap ionization time; (2) monoisotopic precursor selection; (3) number of MS/MS events; (4) capillary temperature; and (5) tube lens voltage. Minimal influence on the proteome coverage was observed for the remaining four parameters (dynamic exclusion duration, resolving power, minimum count threshold to trigger a MS/MS event, and normalized collision energy). The DOE approach represents a time- and cost-effective method for empirically optimizing MS-based proteomics workflows including sample preparation, LC conditions, and multiple instrument platforms.}, number={4}, journal={JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY}, author={Andrews, Genna L. and Dean, Ralph A. and Hawkridge, Adam M. and Muddiman, David C.}, year={2011}, month={Apr}, pages={773–783} }
 @article{gokce_andrews_dean_muddiman_2011, title={Increasing proteome coverage with offline RP HPLC coupled to online RP nanoLC-MS}, volume={879}, ISSN={["1873-376X"]}, DOI={10.1016/j.jchromb.2011.01.032}, abstractNote={Fractionation prior to mass spectrometry is an indispensable step in proteomics. In this paper we report the success of performing offline reversed phase high pressure liquid chromatography (HPLC) fractionation on a C18 2.0 mm×150 mm column at the peptide level with microliter per minute flow rates prior to online nano-flow reversed phase liquid chromatography mass spectrometry (nanoLC-MS) using the well-studied fungus Saccharomyces cerevisiae. A C18 75 μm×150 mm column was used online and the online elution gradients for each fraction were adjusted in order to obtain well resolved separation. Comparing this method directly to only performing nanoLC-MS we observed a 61.6% increase in the number of identified proteins. At a 1% false discovery rate 1028 proteins were identified using two dimensions of RPLC versus 636 proteins identified in a single nano-flow separation. The majority of proteins identified by one dimension of nano-LC were present in the proteins identified in our two dimensional strategy. Although increasing analysis time, this non-orthogonal and facile pre-fractionation method affords a more comprehensive examination of the proteome.}, number={9-10}, journal={JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES}, author={Gokce, Emine and Andrews, Genna L. and Dean, Ralph A. and Muddiman, David C.}, year={2011}, month={Mar}, pages={610–614} }
 @article{andrews_simons_young_hawkridge_muddiman_2011, title={Performance Characteristics of a New Hybrid Quadrupole Time-of-Flight Tandem Mass Spectrometer (TripleTOF 5600)}, volume={83}, ISSN={["0003-2700"]}, DOI={10.1021/ac200812d}, abstractNote={The TripleTOF 5600 System, a hybrid quadrupole time-of-flight mass spectrometer, was evaluated to explore the key figures of merit in generating peptide and protein identifications that included spectral acquisition rates, data quality, proteome coverage, and biological depth. Employing a Saccharomyces cerevisiae tryptic digest, careful consideration of several performance features demonstrated that the speed of the TripleTOF contributed most to the resultant data. The TripleTOF system was operated with 8, 20, and 50 MS/MS events in an effort to compare with other MS technologies and to demonstrate the abilities of the instrument platform.}, number={13}, journal={ANALYTICAL CHEMISTRY}, author={Andrews, Genna L. and Simons, Brigitte L. and Young, J. Bryce and Hawkridge, Adam M. and Muddiman, David C.}, year={2011}, month={Jul}, pages={5442–5446} }
 @article{muddiman_andrews_lewis_notey_kelly_2010, title={Part I: characterization of the extracellular proteome of the extreme thermophile Caldicellulosiruptor saccharolyticus by GeLC-MS2}, volume={398}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-010-3955-6}, abstractNote={The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms, no doubt harbors technologically important enzymes and other thermostable biomolecules that, to date, have been characterized only to a limited extent. In the first of a two-part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS2 analysis and prediction servers suggested probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments, all including the C18 bead technique, demonstrated that two levels of sample purification were necessary to effectively desalt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described, as enzymatic and putative uncharacterized proteins, anticipate consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion, and 11 contained transmembrane domain stretches suggested by Phobius and transmembrane hidden Markov model. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study, involved acetone precipitation followed by the C18 bead method in which 2.4% (63 proteins) of the predicted proteome was identified, including proteins suggested to have secretion and transmembrane moieties.}, number={1}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Muddiman, David and Andrews, Genna and Lewis, Derrick and Notey, Jaspreet and Kelly, Robert}, year={2010}, month={Sep}, pages={377–389} }
 @article{andrews_lewis_notey_kelly_muddiman_2010, title={Part I: characterization of the extracellular proteome of the extreme thermophile Caldicellulosiruptor saccharolyticus by GeLC-MS2 (vol 398, pg 377, 2010)}, volume={398}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-010-4102-0}, number={4}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Andrews, Genna and Lewis, Derrick and Notey, Jaspreet and Kelly, Robert and Muddiman, David}, year={2010}, month={Oct}, pages={1837–1837} }
 @article{muddiman_andrews_lewis_notey_kelly_2010, title={Part II: defining and quantifying individual and co-cultured intracellular proteomes of two thermophilic microorganisms by GeLC-MS2 and spectral counting}, volume={398}, ISSN={["1618-2642"]}, DOI={10.1007/s00216-010-3929-8}, abstractNote={Probing the intracellular proteome of Thermotoga maritima and Caldicellulosiruptor saccharolyticus in pure and co-culture affords a global investigation into the machinery and mechanisms enduring inside the bacterial thermophilic cell at the time of harvest. The second of a two part study, employing GeLC-MS2 a variety of proteins were confidently identified with <1% false discovery rate, and spectral counts for label-free relative quantification afforded indication of the dynamic proteome as a function of environmental stimuli. Almost 25% of the T. maritima proteome and 10% of the C. saccharolyticus proteome were identified. Through comparison of growth temperatures for T. maritima, a protein associated with chemotaxis was uniquely present in the sample cultivated at the non-optimal growth temperature. It is suspected that movement was induced due to the non-optimal condition as the organism may need to migrate in the culture to locate more nutrients. The inventory of C. saccharolyticus proteins identified in these studies and attributed to spectral counting, demonstrated that two CRISPR-associated proteins had increased expression in the pure culture versus the co-culture. Further focusing on this relationship, a C. saccharolyticus phage-shock protein was identified in the co-culture expanding a scenario that the co-culture had decreased antiviral resistance and accordingly an infection-related protein was present. Alterations in growth conditions of these bacterial thermophilic microorganisms offer a glimpse into the intricacy of microbial behavior and interaction.}, number={1}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Muddiman, David and Andrews, Genna and Lewis, Derrick and Notey, Jaspreet and Kelly, Robert}, year={2010}, month={Sep}, pages={391–404} }
 @article{andrews_lewis_notey_kelly_muddiman_2010, title={Part II: defining and quantifying individual and co-cultured intracellular proteomes of two thermophilic microorganisms by GeLC-MS2 and spectral counting (vol 398, pg 391, 2010)}, volume={398}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-010-4050-8}, number={4}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Andrews, Genna and Lewis, Derrick and Notey, Jaspreet and Kelly, Robert and Muddiman, David}, year={2010}, month={Oct}, pages={1839–1839} }
 @article{andrews_shuford_burnett_hawkridge_muddiman_2009, title={Coupling of a vented column with splitless nanoRPLC-ESI-MS for the improved separation and detection of brain natriuretic peptide-32 and its proteolytic peptides}, volume={877}, ISSN={1570-0232}, url={http://dx.doi.org/10.1016/j.jchromb.2009.02.040}, DOI={10.1016/j.jchromb.2009.02.040}, abstractNote={The circulating concentration of a biomarker for congestive heart failure, Brain (B-type) Natriuretic Peptide (BNP-32), is measured using ELISA based assays in order to rapidly diagnose and monitor disease progression. The lack of molecular specificity afforded by these assays has recently come into question as emerging studies indicate there are potentially multiple heterogeneous forms of BNP in circulation with immunoreactive capabilities. In order to better understand the molecular biology of BNP-32 as it relates to congestive heart failure, it would thus be advantageous to use a detection platform such as Fourier transform ion cyclotron resonance mass spectrometry. This high resolving power mass spectrometer can provide unparalleled molecular specificity and can facilitate identification and characterization of the various molecular forms across all disease states. Unfortunately, BNP circulates at low concentrations (as low as 3fmol/mL). Thus, it will require a collaborative effort from a number of orthogonal front-end technologies to overcome the disconnect between the practical detection limits of this instrument platform and the physiological levels of BNP-32 and its alternative molecular forms. Herein, we begin optimization of these front-end techniques by first enhancing the conditions for online nanoLC-ESI-MS separations of BNP-32 and its proteolytic fragments. Through extensive analysis of various chromatographic parameters we determined that Michrom Magic C8 stationary phase used in conjunction with a continuous, vented column configuration provided advanced chromatographic performance for the nano-flow separations involving intact BNP-32 and its associated tryptic peptides. Furthermore, conditions for the tryptic digestion of BNP-32 were also studied. We demonstrate that the use of free cysteine as an alkylation quenching agent and a secondary digestion within the digestion scheme can provide targeted tryptic peptides with increased abundances. Combined, these data will serve to further augment the detection of BNP-32 by LC-MS.}, number={10}, journal={Journal of Chromatography B}, publisher={Elsevier BV}, author={Andrews, Genna L. and Shuford, Christopher M. and Burnett, John C., Jr. and Hawkridge, Adam M. and Muddiman, David C.}, year={2009}, month={Apr}, pages={948–954} }