P1. Real-time LC-IR with total Removal of Solvent
Boris Boumajny, Tom Kearney, and Sidney Bourne, Spectra Analysis,
Inc.
Corresponding Author Email Address:
LC-IR instrumentation has been under development for many years, with mixed results. Published attempts to build a viable interface to remove the LC solvent, and to produce quality IR spectra from LC eluants, date from the late 1970’s. But a good, general purpose LC-IR interface has been elusive, and although a number of techniques have shown promise, commercial instruments available to date have been quite limited in their applicability. A new instrument, not yet introduced to the market as of the writing of this abstract, is our latest step forward in the development of LC-IR. We are still characterizing the performance, and components of that characterization make up the subject of this poster. We are able to demonstrate total solvent removal from a wide reverse-phase gradient run (5% to 100% organic) with no change in the operational setpoints of the system. We compare the IR spectra of several of the eluants with those from commercial IR libraries, to note the differences that can be expected due to differences in phase or crystallinity. Control of the pH in the LC can impact the resulting spectrum, since the various salt forms of ionizable compounds show spectral differences relative to the neutral compounds. This poster will show the current state of the LC-IR experiment.
P2. Comparison of HPLC and SFC Chiral Separations on Coated Amylose and Cellulose 3,5-Dimethylphenyl Carbamate Columns and Next-Generation Immobilized Columns
Kelly A Nadeau – Process
Analytical Sciences Amgen, Inc.
Corresponding Author Email Address:
Within the pharmaceutical industry, coated polysaccharide-based stationary phase columns are commonly used for chiral analyses. Amylose 3,5-dimethylphenyl carbamate and cellulose 3,5-dimethylphenyl carbamate are two of the most commonly used chiral stationary phases. The main limitation with this type of coated column is its incompatability with such solvents as tetrahydrofuran, methyl tert-butyl ether, dichloromethane and ethyl acetate. Building on the success of these columns, a more robust line of columns was introduced to overcome this solvent issue. Immobilized amylose and cellulose 3,5-dimethylphenyl carbamate stationary phases allow greater freedom in solvent choices that were previously damaging to the coated stationary phases. As the immobilized columns are relatively new to the market, the similiarities and differences between these columns and the previous coated columns have not been thoroughly investigated. In this study, the coated phases are compared with the corresponding immobilized new generation columns. The properties of these columns are probed with acidic, basic and neutral compounds using both high pressure liquid chromatography (HPLC) and supercritical fluid chromatography (SFC).
P3. Long, Mixed-Column Nanobore Chromatography for Complex Proteomic Analysis
Christopher J. Toher, Adam W. Perala, Carla J. Marshall-Waggett, Gary A. Valaskovic New Objective, Inc., Woburn, MA
Corresponding Author Email Address:
Complex proteomic digests can require a combination of either multi-dimensional biphasic packed beds or longer reverse-phase beds (greater than 15 cm) to achieve adequate separation. Multi-dimensional and extended-length columns are time-consuming and expensive to produce; column failure of one section of the packed bed requires replacement of the entire column. An attractive alternative to a single biphasic or long-bed column is based on efficient column-to-column coupling, as employed with conventional-bore high performance liquid chromatography. Such connections in nanobore LC are enabled by coupling shorter-bed columns via optically-clear high pressure zero-dead-volume (ZDV) unions. This approach facilitates extended-column configurations and column swap-out during system maintenance. Using two clear ZDV unions to achieve flush connections to column bed termini, two conventional 10cm bed nanobore columns were coupled and connected to the bed terminus of a 10cm nanobore column with integrally fritted tip. Analytical performance of this extended bed column was then compared with a single 30cm-bed nanobore column with integrally fritted tip. Chromatographic data from single and extended-bed configurations were nearly identical with negligible peak-tailing and resolution loss. The first section of the column is readily removed, replaced, and effectively integrates guard column performance into the analytical column bed.
P4. Human Serum and Plasma Protein Depletion – Novel High Capacity Affinity Column for the Removal of the ‘Top 14’ Abundant Proteins
Peter Mrozinski, Nina Zolotarjova, Haiying Chen - Agilent Technologies, Inc.
Corresponding Author Email Address:
The serum and plasma proteome are desirable biological samples due to their accessibility and representative complexity. There is great hope that the investigation of these samples will lead to the discovery of new protein markers for disease diagnosis and therapeutic monitoring and novel drug targets. The tremendous complexity of the plasma proteome presents extreme analytical challenges in proteome characterization.
Depletion of high-abundant proteins in serum and plasma has become routine and an accepted technique. These high-abundant protein components interfere with identification and characterization of important low-abundant proteins by limiting the dynamic range for mass spectral and electrophoretic analyses.
We are presenting the results on a new device for the specific depletion of the 14 high abundant proteins from serum and plasma. By depleting these 14 high-abundant proteins we are removing ~95% of the total protein mass. This depletion column has the same specificity, reproducibility, and high capacity as the industry standard Top-7 device. The device depletes the 14 targeted-proteins with robust performance for over 200 runs and excellent depletion efficiency as determined by ELISA.
The depletion results in an improved dynamic range for proteomic analysis. Furthermore, the removal of the high-abundant proteins improves loading capacity on 2DGE and LC/MS, which simplifies a complex system in the goal of discovering biomarkers.
P5. Evaluation of Precision with Ultra-Fast LC Conditions
Masatoshi Takahashi and William Hedgepeth, Shimadzu Scientific Instruments, Inc.
Corresponding Author Email Address:
There is a growing trend toward the use of smaller (< 3 um) particle size columns to reduce analysis time and increase sample throughput. The use of sub-2um particle size columns has required specialized instrumentation for the high backpressures generated by these columns. There is concern that high backpressure is associated with increased system maintenance and also leads to heat generation within the column, which can adversely affect reproducibility results. The use of a steep gradient over a short run time may also provide poor retention time reproducibility if the pump resolution is not high enough. Recently, a novel 2.2 um particle size column was introduced that allows efficiencies similar to sub-2um columns, but at greatly reduced system backpressures. The use of this column with a conventional LC system that features high resolution pumps (3nL/step) and a high-speed autosampler (10 second cycle time) now allows higher throughput to be obtained with backpressures similar to traditional HPLC conditions. Retention time and peak area reproducibility results for an ultra-fast LC separation will be presented.
P6. Increasing the Speed of Fast LC using Conventional HPLC Gradient Systems
Masatoshi Takahashi, Masayuki Nishimura, William Hedgepeth, Shimadzu Scientific Instruments Inc, Columbia, MD, USA
Corresponding Author Email Address:
Today’s demand for increased efficiency has highlighted the important technical challenge of providing faster separation in HPLC. The two most promising approaches to increasing the number of samples that can be analyzed per day include: 1. Use of smaller particle packing material 2. Separation under elevated temperature The common goal of the two is to reduce HETP and to increase the column performance per column length in a high flow-rate region. The highest throughput is achieved by reducing chromatographic run time, and by speeding the entire cycle time of the analysis. The entire cycle time consists of column equilibration time for gradient analysis, as well as autosampler injection cycle time. As analysis times become shorter, column equilibration times and autosampler injection speeds determine limits for cycle times that can be achieved.
P7. Analysis of Transporter Function in Native Synaptic Vesicles Using a Novel Electrophysiological Technology
Ulrich Pehl, Petr Obrdlik, Kerstin Diekert, Inga Barth, Christine Keipert, Catrin Steensen, Nicole Böhm, Wolf Berger, Maarten Ruitenberg, Walter Volknandt*, Béla Kelety, IonGate Biosciences GmbH, Industriepark Hoechst D528, 65926 Frankfurt/Main Germany
Corresponding Author Email Address:
IonGate Biosciences (Frankfurt/Germany www.iongate.de) has developed a first-of-its kind instrument based on an entirely new cell-free electrophysiology principle tailored for biological transporters, ion pumps and ligand-gated ion channels. The central element of the instrument is an electrochemical biosensor with a specially treated gold surface designed to specifically adsorb transporter-containing membrane targets. The membrane components self-assemble on the gold surface to form a large number of small vesicles doped with transporter molecules. These solid supported membranes (SSMs) are highly stable and enclose isolated small volumes of buffer from the bulk solution. The SSM acts as a carrier for the membrane fragments, and in parallel as a high-capacitance, low-conductance electrode. Via rapid solution exchange, substrate ions are moved by energy (ATP)- or gradient driven transport processes out or into the vesicle cell and trigger a capacitive charging current on the underlying sensor surface. The SURFE2R technology allows direct electric measurements of transporters in plasma membranes as well as in intracellular compartments. To investigate the function of intracellular transporters and channels of mammalian synapse, we purified synaptic vesicles from rat brain. Two vesicular transport systems were analyzed: the synaptic V-type ATPase and vesicular chloride channels. The V-ATPase builds an electrochemical proton gradient across the vesicular membrane serving as the driving force for vesicular transporters. Chloride serves as a counterion for protons and is important for the building of the proton gradient. Characterization of the synaptic vesicles is ongoing with the final goal to identify further synaptic transporters.
P8. A New Generation of MEMS Liquid Flow Sensors for Drug Delivery, Medical Diagnostics and Separation Technologies
Ohlan Silpachai, Carsten Haber, Michael Karst, Ulf Kanne, Sensirion Inc.
Corresponding Author Email Address:
Accurate flow measurements are playing an important role in many applications for analytical instrumentation, process technology and separations. A growing field of interest is capillary LC-MS, and the possibility to introduce a flow-check reference point before sample introduction into the mass-spec instrument. This can be achieved by a miniaturized calorimetric flow sensor based on CMOS technology, permitting noninvasive measurement of flow rates in the range of nanoliters to milliliters per minute. Principle: A miniaturized heat source on the silicon chip introduces thermal power into the medium. For a single measurement, a minute amount of energy of 90–300 µJ is needed. Two thermopiles (T1 and T2) are positioned on the chip symmetrically from a heating element, one upstream and one downstream, each at a distance of a few micrometers. The thermopiles detect slight temperature differences down to 0.002 °C within milliseconds, thus providing basic information about the distribution of the caloric energy in the medium. Flow causes a thermal fractionating of the heat to the downstream temperature sensor and the resulting temperature difference between the two measurement points T1 and T2 generates a precise, measurable signal. This is the fundamental information needed to calculate the actual total flow.
P9. Unique Capabilities of a fast LC/QQQ Mass Spectrometer for Applications in Forensics/Toxicology.
Michael Zumwalt,
Thomas Trainor, Agilent
Technologies,
Corresponding Author Email Address:
A variety of applications are well suited for analysis by liquid chromatography / triple quadrupole mass spectrometry (LC/QQQ) in which sensitivity and acquisition speed are needed. Three applications are highlighted in this work demonstrating different, but unique aspects of the LC/QQQ. For example, for fast analysis and high speed acquisition of drugs-of-abuse compounds in oral fluids, the LC/QQQ is shown to not only have the necessary sensitivity to meet criteria for both SAMHSA and WADA, but able to monitor both the quantitative and qualitative ions for each compound, confirming their presence in the oral fluid matrix using ion ratios. Furthermore, the need for reproducibility is met, even for overlapping peak widths of just a few seconds (ultra-fast HPLC using Agilent 1200 RRLC, column: 1.8 um SB-C18, 2.1 x 50 mm, Agilent 6410 QQQ MS).
A second application involving the LC/QQQ is the analysis of anabolic substance in urine, an area of doping control where the use of electron impact high resolution mass spectrometry has traditionally been the analytical technique of choice. Advantages of using LC/QQQ for this analysis include cost, removes the need for derivatizing the compounds for GC/MS compatibility, and has the sensitivity to meet minimum required performance levels (MRPLs) as required by WADA for doping control. Finally, currently one of the more significant areas of interest in toxicology involves the analysis of immunosuppressant compounds in blood at very low levels and in the relatively complicated matrix. This requires the need for tandem mass spectrometry in the form of a QQQ to selectively remove the interferences of matrix ions with the analytes of interest and to further enhance sensitivity using collisionally induced fragmentation to form product ions specific to the analyte of interest and for subsequent use in quantitation. Because of the levels of toxicity in many of these compounds, the LC/QQQ is necessary for quantitation in the 1 - 10 ng/mL range.
P10. Design of a versatile LC/MS vacuum-pump enclosure that facilitates maintenance while reducing pump noise.
R. Ricker, T. Trainor, Agilent Technologies,
Corresponding Author Email Address:
Today’s busy LC/MS labs are challenged with the routine operation and maintenance of an ever more sophisticated array of instruments – ranging from the basic single-stage MS quadrupole and time of flight (TOF) to multi-stage MS/MS analyzers (ion-trap, triple quadrupole, and QTOF). Common to all mass spectrometers is a rotary vacuum pump (rough pump) that normally sits on the floor below the instrument. Over time, the required frequent maintenance of the rough pumps (visual check of oil levels, oil changes, oil additions, clean-up of oil leaks, etc.) and inherent noise levels can become an annoyance, particularly for operations with multiple LC/MS systems.
This poster describes a second-generation rough pump acoustic
enclosure, with sound deadening insulation and multiple access panels, developed to mitigate all of these issues. To be discussed are several performance tests on the new unit - including determinations of noise reduction and rough pump temperature control. Elevated ambient temperature experiments on the unit showed that, due to the two internal fans and subsequent air flow, the pump oil temperature is kept below the temperature reached by the pump oil in the reference experiment (no enclosure unit), insuring longer oil lifetimes. Further protecting the rough pump are a temperature sensor and audible alarm if temperatures exceed a limit.
A step-on tilting apparatus and full-size removable oil pan allow oil to be effectively changed without lifting or moving the rough pump. With this new enclosure device there is no need to remove or disconnect the rough pump for reading oil level, changing oil, or setting ballast. The G3199B Quiet Cover now provides a solution for a wide variety of LC/MS instruments in the industry utilizing three common rough pump models (BOC Edwards E1M18, E1M28, and E2M28).
P11. Automated Off-line Multidimensional LC Method for Peptides and Proteins
Bas Dolman2, Mark van Gils1,
Robert van Ling2, Evert-Jan Sneekes2, Remco Swart2 1 Dionex
Corporation,
Corresponding Author Email Address:
Azita.Kaffashan@dionex.com
Multidimensional LC separation is used increasingly for the analysis of complex samples in proteomics and pharmaceutical biotechnology to increase resolution. The development of multidimensional LC methods involves optimization of various experimental parameters. Two of the foremost issues that must be considered are (1) an on- or off-line approach, and (2) to perform the separation on the peptide or protein level. Off-line multidimensional LC techniques have several advantages over on-line approaches: (1) more efficient separations, (2) higher flexibility with respect to column dimensions and mobile phase selection, and (3) ability to perform reinjection of fractions. Performing protein digestion inevitably makes the sample more complex, but allows for high throughput tandem MS detection and identification. Here we present an LC instrument designed for fully automated multidimensional LC, with both dimensions in the µL/min flow range. The micro fractionation option (µFC) of the WPS-3000 allows both injection and fractionation. The instrument injection and fractionation performance is discussed. An application of fully automated 2-D LC of peptides is presented.
P12. Accelerated Lys-C Digestion for Quick Oxidation Measurement of Protein Pharmaceuticals
Li Zang,
Tyler Carlage, Yelena Lyubarskaya, Analytical Development Department, BiogenIdec,
Corresponding Author Email Address:
Oxidation is one of the modifications monitored for protein pharmaceuticals during their development. For proteins containing multiple oxidation sites, LC-MS peptide mapping is usually the method used to measure the oxidation on the peptide level. Peptide mapping of large proteins is typically a lengthy procedure including overnight enzymatic digestion and long chromatographic separation. In order to shorten this procedure and improve the throughput of oxidation assay, accelerated enzymatic digestion (Lys-C) has been developed. Two digestion procedures have been compared: digestion under microwave radiation and digestion in the presence of acetonitrile in aqueous buffer. The Lys-C digestion was found to proceed much faster in over 30% acetonitrile in aqueous buffer, while microwave radiation did not appear to improve the digestion compared to routine Lys-C digestion in aqueous buffer. Digestion time and temperature were next evaluated and 1-h digestion at 25°C in 30% acetonitrile was selected as the optimized accelerated condition for Lys-C digestion. The obtained protein digest was 10-fold diluted with water for analysis by a flow-injection LC-MS method developed in our laboratory. This method uses a short on-line desalting cartridge in place of a separation column. The combination of the accelerated Lys-C digestion with the fast flow-injection LC-MS analysis allows for protein oxidation to be measured within 90 min, which provides a much shorter turn-around time for the assay.
P13. A multi-level depletion strategy enabling the identification of sialyated glycoproteins in plasma.
Christina Orazine, Marina Hincapie, William
S. Hancock, Barnett Institute and Department of Chemistry and Chemical Biology,
Northeastern University,
Corresponding Author Email Address:
The large dynamic range of proteins in plasma requires the development of methods of prefractionation aimed at reducing sample complexity. Recently our lab introduced the use of Multi Lectin Affinity Chromatography (M-LAC) for the broad capture of serum/plasma glycoproteins. We now present an automated two-dimensional affinity HPLC strategy for the enrichment of sialyated glycoproteins in human plasma. Changes in sialic acid are increasingly being recognized as one of the most common post-translational alterations associated with malignant transformation and tumorigenesis. In this study we develop a double depletion strategy for high throughput analysis of this clinically relevant class of proteins.
In the first column the two most abundant plasma proteins (i.e. albumin and Immunoglobulin G) are depleted using POROS™ anti-albumin and Protein A. The flow-through (depleted plasma) is captured directly onto a newly developed POROS™ sialic acid lectin (SAL) column. The eluent from the second column (SAL) in this method contains the non-glycosylated proteins and non-sialyated glycoproteins. Both the flow through and the bound fraction (sialyated glycoproteins) are digested with trypsin, the resultant peptides are further separated and sequenced by nano-flow reversed phase HPLC coupled to a linear ion trap mass spectrometer (LTQ MS). This approach greatly simplifies the plasma glycoproteome, permitting the identification of low abundance sialyated glycoproteins in human plasma. This strategy was used in the proteomic analysis of plasma from prostate cancer patients for the discovery of potential cancer markers, and to monitor proteome-wide glycosylation changes related to this cancer.
P14. A Technique to save time in the evaporation step after preparative HPLC fraction collection
J.M. Stevens, M. Crawford, T. Hegeman, L. Roenneburg, Gilson Inc.
Corresponding Author Email Address:
Evaporation of collected fractions is a major bottleneck in the production of dry samples of purified compounds for small molecule drug discovery. This paper explores on line and off line approaches to speed the evaopration process by exchanging the elution solvent with pure ACN using SPE.
P15. High-throughput methods for discovery of lower abundance biomarkers using a combinatorial library of peptide ligands and SELDI-TOF MS
Ronald Woodbury, Amanda Bulman, Kedron Hooker, John Deuso, and Diane McCarthy, Life Science Group, Bio-Rad Laboratories, Inc.
Corresponding Author Email Address:
Biomarker discovery from human biological samples is often limited by the availability of sufficient sample volumes and is complicated by the wide dynamic range of the human proteome. The challenge of a broad dynamic range is especially significant for serum, where approximately a dozen proteins comprise over 90% of the total serum protein concentration, but this is also true for other sample types. Although many fractionation methodologies have been developed to address this challenge, most are low throughput and tend to dilute the sample, making downstream analysis more challenging. A novel fractionation method utilizing a peptide ligand bead library*, coupled with SELDI-TOF MS analysis of each fraction, demonstrated a two to three fold increase in the number of unique peaks detected in several biological fluids. Treatment of samples with the peptide ligand bead library reduces the dynamic range of proteomes, simultaneously diluting high abundance proteins and concentrating low abundance proteins, such that the eluted materials consist of a significantly lower amount of total protein representing a higher diversity of species.
It has been previously demonstrated [Thulasiraman, Electrophoresis (2005) 26: 3561-357; Castagna, J Proteome Res. (2005) 4(6):1917-30] that this ligand library bead technology can greatly enrich low abundance proteins in serum and urine. Recent work has focused on developing high throughput, highly reproducible methods for enrichment of low abundance proteins using this peptide ligand library with clinically relevant volumes of human samples. SELDI was selected for analysis of the library bead fractions because the chromatographic nature of the SELDI surface allows for direct, rapid TOF-MS analysis of bead eluates in a robust, high-throughput manner. Furthermore, since SELDI analyses require very low sample volumes, each fraction can be profiled on multiple ProteinChip Array surfaces (e.g. reverse phase, anion and cation exchange, metal affinity) to further increase the number of detectable species. Robust, high throughput methods have been developed for the analysis of diverse sample types, including serum/plasma, urine, subcellular lysates, and cervical lavage.
P16. Determination of the Protein Concentration and Product Quality in Conditioned Media by Two-Dimensional Chromatography on-Line with Mass Spectrometry (2D-LC/MS)
Damian Houde, Zoran Sosic, Steven Berkowitz, Yelena Lyubarskaya and Rohin Mhatre (Analytical Development, Biogen Idec)
Corresponding Author Email Address:
The physicochemical characterization of recombinant protein biopharmaceuticals plays a critical role not only for product release but also during the biopharmaceutical process development. The integration of different analytical methodologies enables identification and characterization of complex biologics in order to meet requirements of Food and Drug Administration (FDA) for a consistent drug production process and product quality. However, the ability to identify different sources of a recombinant protein heterogeneity in complex sample matrices in a timely and quantitative manner remains a significant challenge.
In this work, we describe a 2-dimensional liquid chromatography approach with on line UV and mass spectrometric (MS) detection to assess protein titer and quality of a recombinant glycoprotein in conditioned media samples. This rapid and automated analytical approach provides important information to enable timely and efficient control and optimization of protein production process. The method is based on the use of a home built 2D-LC/MS system that employs ion exchange chromatography followed by reverse-phase chromatography with UV and MS detection. The set-up includes a switching valve to redirect the column flow from ion-exchange column onto a reverse-phase column for further separation and protein titer quantitation by UV absorbance at 280 nm. An additional switching valve has been used to couple 2D-LC system with an on-line electrospray ionization quadrupole time of flight mass spectrometer (ESI-qToF-MS) for analysis of protein glycoforms. Details of the assay development and performance will be discussed.
P17. High-Speed Amino Acid Analysis on 1.8 Micron Reversed-phase Columns
Cliff Woodward, John Henderson and Todd Wielgos* Agilent Technologies and *Baxter Healthcare
Corresponding Author Email Address:
In life science research, the analysis of amino acids is an important
qualitative and quantitative technique for characterizing protein hydrolysates. Since the original days of Stein and
P18. Novel Planar Differential Mobility Spectrometry (DMS) as effective pre-filter for APCI-MS
Evgeny V Krylov; Stephen L Coy; Raanan A Miller; Erkinjon G Nazarov, Sionex Corp.,
Corresponding Author Email Address:
Novel Aspect: Efficient low-loss planar DMS-MS system improves S/N, operates in transparent or filtering modes, and provides new information on chemical kinetics.
Differential mobility based
pre-filter improves signal noise ratio in mass spectrometry by reducing
chemical noise, and shortens analysis time by replacing time-consuming
pre-separation methods such as LC or SPE. In this work, we describe new designs
for DMS-MS interfaces and present experimental data with electrospray
and radioactive ion sources. Applications are shown that cover a molecular
weight range from several hundred to more than a thousand Da, and the technique is applicable to proteins and
peptides with molecular weights of several thousand
We describe miniature linear flow planar DMS pre-filter for analytical applications. This planar, linear-flow, design has several advantages over the familiar cylindrical design, and over other planar configurations:
- planar DMS selectivity is 2-4 times higher than that of cylindrical designs;
- the time delay within the planar DMS unit is only 1-2 ms, minimizing unwanted chemical reactions at ambient pressure conditions;
- positive and negative ions are transmitted and filtered in DMS simultaneously;
- planar DMS can be switched between a transparent and a filtering mode, which provide a quick test of the S/N improvement and ion selectivity;
- transmission and filtering efficiency remains high for high molecular weight ions, in contrast to cylindrical designs.
Several small peptides with additional interfering compounds have been used as test samples for the electrospray planar DMS pre-filter. We present results showing that analyte ion fractions are frequently increased by factors of 50 or more by DMS filtration. In cases where different charge states appear at nearly the same m/z values, DMS was capable of suppressing the unwanted charge states completely. This is possible because ion mobility dependence on molecular conformation, cross-sections and on charge state is non-linear, quite different from the simple m/z dependence of mass spectrometry. Similar results were also obtained for caffeine as a minor component in PEG mixtures of similar molecular weight.
Several molecular cations and anions generated by 63Ni ionization have been used to test the variable temperature/pressure DMS-MS interface. Because planar DMS is used at higher field strengths than cylindrical designs, we have been able to observe collision-induced dissociation (CID) processes in several ions and ion clusters as a function of temperature and mean collision energy, These include the loss of HO2 from oxygen anion clustered with methyl salicylate and the loss of F atom from SF6 anion, among others.
P19. BUDSS: A Software Shell for automated MS Data Processing and Management
Yang Su1,5, Sequin Huang1,3,6, Hua Huang1,3,7, David H. Perlman1,3, Catherine E. Costello1,2,4, Mark E. McComb1,3,1. Cardiovascular Proteomics Center, 2.Mass Spectrometry Resource, 3.Department of Medicine, 4.Department of Biochemistry, Boston University School of Medicine, 5.Bioinformatics Program, Boston University, Boston, MA, 6.Present address: Waters Corp, Milford, MA, 7. Present address: Allergan Inc. Irvine, CA.
Corresponding Author Email Address:
Introduction: BUDSS (Boston University Database Search Shell) is a software shell designed to aid processing and management of various MS data files. For data processing, it allows for the conversion of different MS data types to the formats supported by several search engines and the use of multiple web-based search engines (Mascot, MS-Fit and BUPID) for protein identification. Both data conversion and database search submission can be run in a batch mode to handle large amounts of data with little manual intervention. For data management, BUDSS supports multiple users or projects and organizes data and settings individually for each user or project. Finally, all these functions are accessible through a user-friendly graphical interface.
Methods: BUDSS was developed using Microsoft Visual Basic 6.0. To add support for the mzXML and mzData formats, we used MSXML 4.0 as an XML parser and built a visual C++ library to decode Base64 encoded peak list data in the mzXML and mzData files. As an example demonstrating the functionality of BUDSS, albumin-depleted plasma samples were separated using a Beckman PF2DTM system, trypsin digested and analyzed with a Bruker ReflexTM IV MALDI-TOF mass spectrometer. Raw MS data files were processed first by MoverZTM to yield peak list files in Excel format. BUDSS was then used to covert them into MGF formats and submit converted files to different database search engines. Multiple search parameters were employed. Comparative results were obtained via LC-MS/MS of the same samples.
Results: To explore the impact of different search engines and search parameter settings on search results, we performed PMF searches using BUDSS on a series of MALDI mass spectra from 2D-LC fractions. Three web-based search engines (Mascot, MS-Fit and BUPID) were used; each with nine different parameter settings. Variability between these settings was introduced by changing the taxonomy, number of missed cleavage sites and mass tolerance. For each search setting, we created a unique project within BUDSS and stored corresponding search parameters. Database searches were then run automatically in batch mode with the search parameters automatically applied. Preliminary results from Mascot revealed that MALDI PMF results agreed with definitive identification obtained from LC-MS/MS the majority of the time, but MALDI results were usually limited to one or two proteins in a particular fraction. Additionally, we observed that when Mascot results were consistent across different search settings, then the consensus protein was most likely the true positive. On the contrary, when the search results were different dependent on the parameters used then the occasional significant result may not be evident of positive protein identification.
Conclusions: BUDSS is an easy-to-use graphical interface for performing MS data processing and management and allows for database searching using multiple search engines with multiple search parameters.
Acknowledgements: This project was funded by NHLBI contract N01 HV-28178.
P20. Coupling Protein HPLC to MALDI-TOF-MS: Exploration of an On-Target Device for One-Pot Sample Preparation from Fraction Collection to Peptide Mass Fingerprint Analysis
David H. Perlman,
Hua Huang, Catherine E. Costello, and Mark E. McComb Cardiovascular
Large-scale mass spectrometry-based proteomic analyses require high-throughput upfront separations and sample preparation techniques. Multidimensional protein chromatography offers an attractive alternative to traditional labor-intensive gel-based proteomic separations because of its potential for increased automation. Unfortunately, liquid separations divide the original proteomic mixtures into numerous discrete samples, each of which may require several steps of down-stream sample manipulation, such as fraction collection, buffer exchange, protease digestion, peptide desalting, and, in the case of MALDI-MS, matrix and analyte cocrystallization on target. These multiple steps of time-consuming sample handling can lead to high overall analysis costs and can result in sample contamination and loss. Although robotic liquid-handling devices can facilitate these steps and reduce analyst/sample contact, they remain prototypic and expensive. In order to overcome some of these sample processing hurdles, we explored the use of a novel, inexpensive, one-piece elastomeric device, the BD™ MALDI sample concentrator, that affixes to a MALDI target to create a prestructured 96-well sample array on the target surface, which accommodates a high solution volume (ca. 200mL), thereby enabling on-target processing of samples for MALDI-TOF-MS. We investigated several factors that influence MALDI sample preparation: choice of matrix, solution volume, solution organic composition, solution drying rates and matrix/analyte co-crystallization methods. Furthermore, we have developed methodologies to process large volume fractions from high-flow HPLC (e.g., 1 ml/min) by collecting them directly into the elastomeric device and then subjecting them to sequential on-target sample concentration, buffer exchange, digestion, desalting, and matrix/analyte cocrystallization for MALDI-MS analyses. We demonstrate that this methodology enables the rapid digestion and analysis of low amounts of proteins and that it is effective in the characterization of an HPLC-fractionated protein mixture by MALDI-TOF MS followed by peptide mass fingerprinting (PMF). These methodologies using this simple, on-target elastomeric device show promise for streamlining the sample preparation process from protein HPLC to MALDI-MS PMF analysis.
P21. Fast HPLC Analysis of Additives Extracted from Polymer Formulations – Accurate Method Transfer to Rapid Resolution LC Methoods
Michael Woodman1, Chunxiao Wang2, Wei Luan2, Thomas Trainor1
1 Agilent Technologies, 2850 Centerville Road, Wilmington, Delaware 19808 USA 2 Agilent Technologies China, 412 Ying Lun Road;Pu Dong;Shanghai 200131;CHINA
Corresponding Author Email Address:
Traditional HPLC methods developed on 5 um particle columns are often good candidates for modernization by using shorter and smaller diameter columns packed with sub 2-um particles on instruments that can operate at higher pressures. The potential benefits include reduced analysis time and solvent consumption, improved sensitivity and greater compatibility with mass spectrometers.
In our survey of well-established methods that could benefit from these translation principles, we selected three significant ASTM methods, associated with the analysis of polymer additives, as candidates for this study. Cumulatively, ASTM methods D1996, D5815-95 and D6042-04 analyze the extracts of various polymer matrices, via reversed phase gradient elution HPLC with UV detection, for a large collective group of analytes (BHT, BHEB, Isonox-129, erucamide slip, Irganox-1010, Irganox-1076, Vitamin E, Irgafos 168, Irganox 3114) and are used as the model for method translation principles described in the present work.
For gradient elution
separations, successful method conversion to smaller columns requires that the
gradient slope be preserved. We can express the gradient as %slope =
(End%-Start%)/#column volumes. 
A
large gradient slope yields fast gradients with minimal resolution, while lower
slopes give higher resolution at the expense of solvent consumption and reduced
sensitivity. The user is free to control gradient slope by altering gradient
time and/or gradient flow rate. Longer analysis time may also
result unless the gradient slope is reduced by increasing the flow rate, within
acceptable operating pressure ranges, rather than by increasing the gradient
time.
P22. Differential proteomic characterization of B cell proliferative states: analysis of tumor-specific and proliferation-specific proteomes in normal and malignant B cells
Paul
B. Romesser1, David H.
Perlman2, Anupama Sinha1,
Mark E. McComb2, Douglas V. Faller1, Catherine E.
Costello2 and Gerald V. Denis1,1.
Corresponding Author Email Address:
Novel aspect.
Translational proteomics, to provide frameworks for human study: identifying individualized DLCL biomarkers.
Introduction: The dual bromodomain protein Brd2 is closely related to the basal transcription factor TAFII250, which is essential for cyclin A transactivation and mammalian cell cycle progression. In transgenic (Tg) mice, constitutive lymphoid expression of Brd2 causes diffuse large B cell lymphoma (DLCL), which represents most diagnosed human non-Hodgkin’s lymphoma (NHL) cases, in part through upregulation of the cyclin A locus. In an effort to understand the mechanism underlying Brd2-driven B cell lymphoma and how it differs from proliferating or resting B cells, we undertook 2D-PAGE-based comparative proteomic analyses of Tg lymphoma in relation to proliferating and resting B cells. We hypothesized that, by analogy to our transcriptional profiling results, we would deduce a malignancy-specific signature distinct from normal proliferation.
Method: We used 2D-PAGE separation of murine nuclear extracts subjected to size exclusion
chromatography, purified from normal resting B cells, normal proliferating B
cells, and Tg malignant B
cells, to define 2D reference maps. All cells were derived from syngeneic mice, thus eliminating genetic variance. Resolved,
stained and quantitated protein spots were excised
and digested with trypsin. Peptides were desalted and
subjected to MALDI-TOF MS and peptide mass fingerprint analyses or to LC-MS and
LC-MS/MS analyses. Mass spectra were analyzed with MoverZ
(Genomic Solutions) or MassLynx (Waters) software.
PMF analysis was conducted using Mascot (Matrix Science) and BUPID (
Preliminary Data: Differential comparison of the 2D gels of protein nuclear extracts from resting, stimulated and Tg lymphomic cell highlighted the distinct proteomic phenotypes of each cell type. Global differences in patterns supported the hypothesis that there exists a basal B cell proteomic profile, a general proliferative proteomic signature that is present in both the lymphomic and stimulated B cells, and, most importantly, a discrete group of lymphomic cell-specific proteins that are unique to the Tg lymphoma. Differential analysis allowed the observation of the absolute presence/absence of novel marker proteins, the up- and down-regulation of other implicated proteins, and detection of protein post-translational modifications that were suggestive of altered protein function. Thus, by subtracting the proliferative proteomic signature from the entire lymphomic-cell proteome we could identify key proteins that may be involved in or markers of lymphomagenesis. Proteins subjected to uniquely lymphoma-specific over-expression included those known to be involved in cell cycle control, energy metabolism, cell structure, and mRNA transcription and splicing control. This study thereby represents a paradigm in personalized medicine applied to the diagnosis of lymphoid malignancies, in which a patient’s particular lymphoma-specific protein signature may be resolved from the patient’s background profile of B cell proliferative proteins.
Conclusions: Our results illustrate that lymphomagenesis is more complex than upregulated or dysregulated normal cellular proliferation; it involves additional expression changes in many proteins, several of which were not previously linked to lymphoma. It demonstrates a realistic model of personalized disease diagnosis in which a patient’s B cell ex-vivo proliferative signature can be defined for baseline comparison to suspected new or recurrent lymphomas. Moreover, by shedding light on the protein mechanics particular to proliferation and carcinogenesis, our results may lead to identification of novel targets for chemotherapeutic intervention.
Acknowledgements: ACS grant RSG-0507201, NIH grants P41-RR10888, S10-RR15942, CA075107, CA102889, NHLBI contract N01-HV-28178
P23. Electrophoretic Analyses of Proteins and Peptides Isolated From Cortical Bone Using a Pressure Cycling Technology
Gary B. Smejkal, David C. Muddiman*, Mary
H. Schweitzer*, Timothy S. Collier*, and
Corresponding Author Email Address:
Comprehensive proteomic analysis of tissues is only possible when the total protein constituency of cells and extracellular matrices is effectively isolated. The efficiency of sample preparation is therefore a critical component of the analytical process and is essential to reliable proteomic analyses. Cortical bone is an extensively calcified osseous tissue and represents a specific challenge in sample preparation. Historically, bone requires prolonged acid demineralization over several days to enable complete penetration of histochemical reagents to cellular components. Mammalian, avian, and reptilian samples were used to model protein and peptide release from compact bone. Formic, acetic, and hydrochloric acid were evaluated as demineralization reagents prior to the extraction of proteins from bone using a pressure cycling technology (PCT) in which the sample was rapidly cycled 80 times between 35,000 psi and atmospheric pressure. Differences in proteins and peptides released during demineralization with different acids were analyzed by protein assay and electrophoresis and compared to those released by the downstream PCT process. Without prior acid demineralization, PCT yielded more protein than demineralized samples. The ability to extract proteins from bone without prior demineralization offers an important advantage in time savings. Moreover, when working with fossilized bone, where it is assumed that protein remnants will be fragmentary or highly cross-linked, extensive demineralization may further contribute to molecular damage, loss, or modification. Finally, since the carryover of Ca and PO4 from these tissues interferes with IEF, several strategies for the removal of these substances prior to two-dimensional gel electrophoresis were investigated.
P24. ProteinCenter – A Protein Centric Software Package to Accelerate Proteome Research
John Chakel,
Søren Schandorff, Ole Vorm, Morten Bern, Alexandre Podtelejnikov, Proxeon A/S, Staermosegaardsvej
6, DK-5230
Corresponding Author Email Address:
Modern proteomics is facing the challenge of performing high throughput bioinformatics analysis and comparison of large datasets. It has been extremely time consuming and difficult to distinguish known from novel proteins in these data sets without proper annotation and comparison with literature sources. We applied ProteinCenter - a new software tool based on relational database technology - to perform fast and sophisticated large-scale bioinformatics analysis and comparison of proteomes. ProteinCenter integrates public sequence databases to form a comprehensive, consolidated, and consistent master database derived from over 35 million protein records from GenBank, Refseq, EMBL, UniProt, Swiss-Prot, Trembl, PIR, IPI, PDB, Ensembl etc. An important feature of a ProteinCenter is a curated protein sequence index. This allows investigators to control redundancy, include cross-references to a variety of proteins sequence databases, provide stable identifiers for protein records, and track 5 million outdated and deleted sequences. The 8 million unique protein sequences in ProteinCenter are richly annotated by consolidated annotation from public databases together with annotation from internal computational enrichment of the sequence data, including e.g. PFam, trans-membrane domains, and signal peptide predictions. The aggregated ProteinCenter database is updated biweekly. The ProteinCenter interface allows users to import and store data sets (e.g. protein identifications) derived from experimental work or literature studies etc. These can be subsequently be mined, compared to other datasets, and documented in a matter of minutes, using a range of novel methods and filters for handling large proteomics data sets. A comparison of body fluids proteomes, the generation of a global phoshoproteome database, as well as a comparative analysis of four public databases (SwissProt, RefSeq, IPI and NCBInr) will be presented.
P25. Probability-Based Identification of Proteins, Including Post-Translational Modifications and Variants, Using Peptide Mass Fingerprint Data
Tong Weiwei (1,2), Mark E. McComb (1), James West (1), Yang Su (1), David H. Perlman (1), Catherine E. Costello(1), Zhiping
Weng (2), (1) Cardiovascular Proteomics
Center, Boston University School of Medicine, Boston, MA. (2) Department of Biomedical
Engineering,
We present an enhanced version of the probability-based search algorithm, BUPID (Boston University Protein Identifier), to interpret MS data. BUPID is a robust and accurate statistical model to identify proteins and find post-translational modifications. It is optimized to run on a multi-processor server for fast web-based search. We use likelihood-ratio to calculate the probability that a protein is present in the sample. The model compares the probability that a set of peaks in the spectrum is randomly generated, and the probability that they are generated by peptides corresponding to a specific protein. Peak assignment is based on likelihood ratio as opposed to matching peaks and peptides with mass errors less than a cutoff. Final results are ranked by confidence scores calculated from likelihood-ratios. For searching variants and PTMs, the mass spectrum is first matched with unmodified protein sequences. Top-scored sequences in the search result are then used to construct a database with post-translational modifications and variations. The spectrum is re-calibrated and searched against the new database. Database search results for both real and artificial datasets showed that BUPID is accurate and reliable when dealing with various kinds of mass spectra, containing vastly different numbers of peptide peaks and noise peaks.
P26. Tissue-Based Glycomics using Stable Isotope Labels and HILIC - Amide LC-Tandem MS
Alicia M. Hitchcock (1),
Karen E. Yates (2), Catherine E. Costello (1), Joseph Zaia
(1). (1)
Corresponding Author Email Address:
In order to define the functional roles of glycan expression during disease formation it is necessary to determine their patterns of expression as a function of tissue location. The structural heterogeneity of glycosaminoglycans (GAGs) is of interest because it can be related to various biological parameters such as aging and osteoarthritis. The present work applies high resolution capillary amide-80-LC with on-line tandem MS to the analysis of cartilage tissue corresponding to one microgram GAG. A structural analysis of the abundant internal delta unsaturated oligosaccharides, lower abundance linker region, and saturated oligosaccharides in normal and osteoarthritic cartilage samples is described. Isotopic labeling and normal phase-LC tandem MS analysis of GAG oligosaccharides allows for structural characterization and quantification of different glycoforms.
Sulfated GAGs are extracted from papain-digested juvenile bovine and adult human cartilage samples (3 replicates of 1 µg, 3 µg, and 5 µg GAG equivalent) using a streamlined multi-step procedure. Samples were released from residual core protein, passed through a C18 reversed phase solid phase extraction column; ethanol precipitated, enriched using a strong anion exchange column, and desalted using an ethanol precipitation to extract GAGs from the intact cartilage. Oligosaccharides were partially depolymerized followed by derivatization with 2-anthranilic-3,4,5,6-d4 acid. Derivatized cartilage derived GAG samples (cartilage-d4-2AA) were cleaned, spiked with standard CSA-d0-2AA, and subjected to an online capillary amide-80-LC/MS/MS platform. A combination of MS analysis and tandem MS analysis was employed for detailed structural characterization.
1 µg, 3 µg, and 5 µg GAG-equivalent cartilage samples were successfully recovered from the GAG extraction protocol in sufficient quantity to be detected by MS. Nanoscale amide-80 chromatography coupled with online tandem mass spectrometry allows for a greater depth of structural characterization of low quantities of GAG oligosaccharides extracted from juvenile bovine and adult human cartilage tissue. Online amide-80-LC/MS allows for the detection of internal delta unsaturated oligosaccharides ranging in size from disaccharide to dodecasaccharide, and the detection of low abundance, unique product ions including non-reducing end saturated oligosaccharides, under-/over-sulfated oligosaccharides, and linker region oligosaccharides.
Extracted GAG oligosaccharides were also analyzed by amide-80-ESI-LC/MS/MS for a more detailed structural characterization. The increased sensitivity of the amide-80 chromatography allowed for 1 µg, 3 µg, and 5 µg ∆dp4, ∆dp6, and ∆dp8 chains to be quantified using the percent total ion abundances of light and heavy predictive ions containing the reducing end (Y11-, Y32-, and [M-H-SO3]2- for ∆dp4; Y32-, Y53-, and [M-H-SO3]3- for ∆dp6 and Y32-, Y53-, and [M-H-SO3]4- for ∆dp8). Predictive ion contributions from three replicate cartilage samples were then put into a set of three equations and solved for three unknowns that represent the percentage of CSA, CSB, and CSC in each cartilage sample. The results demonstrate that amide-80 LC creates a more sensitive level of detection and allows for the quantification of larger oligosaccharide chains, present in lower quantities. CS extracted from articular cartilage was consistent with expectations, yielding a high 4S:6S GAG ratio in all chain lengths of juvenile bovine cartilage and a low 4S:6S ratio in all chain lengths of adult human cartilage.
This work was supported by NIH grants P41 RR10888 and R01 HL74197. The Esquire 3000 ITMS was donated by Bruker Daltonics, Inc.
P27. Glycation of Interferon-beta-1b and Human Serum Albumin in a Lyophilized Glucose Formulation
Xiaoyang Zheng, Shiaw-Lin Wu, and William S. Hancock Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, 341 Mugar Hall, 360 Huntington Avenue, Boston, Massachusetts 02115 Corresponding Author Email Address:
Purpose: To detect and locate non-enzymatic glycation sites in two different beta-interferon lyophilized formulations.
Methods: Interferon-beta-1b and human serum albumin in both lyophilized glucose formulation and lyophilized mannitol formulation were separated first on a reversed phase C4 column, next digested with trypsin, and then analyzed by a sensitive LC-MS approach. The glycation sites were determined by the accurate mass (FTICR MS) with MS/MS measurements on the corresponding tryptic peptides. The extent of glycation was measured by the ratio of the peak intensity between the glycated and the average value for 3 non-glycated peptides in the same run.
Results: Residues of 18lysine of interferon-beta-1b, and 51lysine, 233lysine, and 545lysine of human serum albumin were more prone to be glycated than other sites in this lyophilized glucose formulation. Residues of 51lysine and 233lysine glycation sites but not 545lysine of human serum albumin are highly accessible to solvent as found in a solution storage study by Lapolla et al. The extent of glycation of both proteins and the number of glycation sites of human serum albumin were increased with the storage time at 25 oC. In total, two glycation sites of interferon beta-1b and 17 glycation sites of human serum albumin were identified in the lyophilized glucose formulation with the storage time at 25 oC for 35 days. Among the 17 glycation sites, the 525lysine of human serum albumin has been found in diabetic patients in vivo by Shaklai et al. As expected, there was no glycation found on both interferon-beta-1b and human serum albumin in the control samples (similar lyophilized formulation but using mannitol instead of glucose).
Conclusions: We have developed a robust, sensitive, accurate and reliable method to detect the existence, location and stability of glycation sites of proteins. This method can be very useful for the development of generic biopharmaceuticals.
P28. Optimized enrichment and detection methodologies for the study of phosphopeptides of the Epidermal Growth Factor Receptor
Amanuel Y. Kehasse1,2, David H. Perlman1, Mark E. McComb1, Ilene Boucher2, Vickery Trinkaus- Randall2, Catherine E. Costello1,2, 1Center for Biological Mass Spectrometry and 2Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118
Corresponding Author Email Address:
Introduction: Epidermal growth factor receptor (EGFR) tyrosine kinase plays an important role in regulating cell growth, proliferation, and migration. Differential phosphorylation of specific tyrosine residues in EGFR in response to diverse external stimuli serves as the key link between these stimuli and the internal signaling pathways that they activate. EGFR phosphosite-specific antibodies have been used as highly sensitive tools to monitor phosphorylation site occupancy, however, they suffer from lack of specificity at concentrations of EGF greater than 0.5 nM. Recently, highly sensitive mass spectrometry based detection strategies have been described to characterize the EGFR tyrosine phosphorylation cascade under a variety of conditions. Here we explore the optimization of sample preparation, phosphopeptide enrichment and detection strategies for the study of EGFR phosphopeptides.
Methods: Human epidermoid carcinoma A431 cells, which over-express EGFR, or the porcine aortic endothelial (PAE) cell line transfected with EGFR expression constructs, or human corneal epithelial cells, were cultured, harvested in the presence of a cocktail of protease and phosphatase inhibitors, and EGFR was subjected to immunoprecipitation. Eluted EGFR, commercially-available purified EGFR (derived from A431 cells), or control cells lacking specific phosphotyrosine residues of EGFR was subjected to SDS-PAGE and in-gel digestion with a panel of proteases. Peptides were eluted and subjected to enrichment by reversed-phase, ion exchange, metal ion affinity, or titanium dioxide affinity chromatography. Phosphopeptides were analyzed by MALDI-TOF MS using a variety of matrices and matrix additives in the positive and negative ion modes.
Preliminary results: We have recovered EGFR from cells in culture with high yield and purity using immunoprecipitation followed by SDS-PAGE. We have explored the use of multiple proteases for in-gel digestion, to target, in particular large tyrosine-containing tryptic peptides that may have been unrepresented in previous MS analyses. Using optimized procedures for recovery from gel, we have subjected the EGFR peptides to various forms of chromatographic enrichment and separation techniques including titanium dioxide chromatography to exploit the differential binding and elution of EGFR phosphopeptides. A panel of matrices and matrix additives are being explored for their capacity to enhance ionization of EGFR phosphopeptides in the positive and negative ion mode of the MALDI-TOF MS. Initial results suggest that significant improvements can be made in sample preparation, enrichment and ionization methodologies in order to maximize the detection of EGFR phosphopeptides.
Acknowledgements: This research is supported by NIH grants P41 RR10888 and S10 RR15942 (to CEC) and R01 EY06000 (to VTR)
P29. Redundant Data Storage and Data Processing Computer Hardware Solution for Mass Spectrometry Laboratories on a Budget
James West, Weiwei Tong, Yang Su, Catherine E. Costello, Mark E. McComb, Cardiovascular Proteomics Center, Boston University School of Medicine, Boston, MA.
Corresponding Author Email Address:
Introduction: Mass spectrometry based proteomics yields large volumes of data in raw and processed forms. Data handling, processing and storage quickly overwhelms the typical capacities of modern PC workstations in a university environment. Recent advances in computer technology have made high performance computing available to individual laboratories. Here we describe infrastructure, hardware and software requirements for the design and implementation of an affordable and easily deployable high performance computing network for proteomics and bioinformatics analyses.
Methods: We explored hardware and software technologies available with respect to the needs of a proteomics laboratory. These included symmetric-multiprocessing (SMP) vs. blade and clustering solutions, AMD vs. Intel chip design, 32- vs. 64-bit design, storage technologies, operating system (OS) interoperability, local and network security policies, secure and virtual LAN, and integration within existing infrastructure.
Results: The resultant high performance compute solution consists of a RAID based data storage array server and two compute servers. The compute servers consist of 4-way AMD Opteron 880 dual-core chips at 2.4 GHz on a Tyan motherboard with 32 GB RAM operating in full SMP mode with 2 TB RAID 5 storage and dual gigabit data ports. BU Linux 4.5 Server Edition (Zodiac) and Windows 2003 Server Enterprise R2 were chosen to fully support software deployment and programming development. The servers host MASCOT (Matrix Science), ProteinLynx Global Server 2.2 (Waters Corporation) and BUPID for data processing. The redundant data storage array server utilized two AMD Opteron 2216 processors (2.4 GHz), 4GB RAM, dual gigabit data ports, with 4 TB of local disk space; RAID 6 double parity, and a separate 40 GB RAID 1 array for Windows 2003 Server R2. Laboratory integration with instrumentation is via virtual LAN and integration via the BUSM infrastructure is via Active Directory. To evaluate system performance we analyzed existing LC-MS/MS data sets. We processed data in identical runs and compared results to those obtained using a Dell Optiplex GX620. Additional benchmarking was by Sandra Lite 2007. Data processing tests showed a 10-fold increase in floating point operations per second (FLOPS) and a 6-fold increase in overall performance for the 4-way system. System and network stability were obtained.
Conclusions: High powered compute solutions with large redundant storage capacities are no longer financially and technically out of reach of most scientific laboratories.
Acknowledgement: This project was funded by NIH NHLBI contract N01-HV-28178.
P30. Maximizing Peak Capacity for High Speed Peptide Mapping on 1.8u particle Columns
Cliff
Woodward, Agilent Technologies,
Corresponding Author Email Address:
In Proteomics the speed of data production is highly important. One of the principal limitations to this speed is the time needed for peptide mapping on conventional 250mm columns with 5u particles. The use of shorter columns enables high speed analysis; and this, coupled with 1.8u particle packings, can produce chromatograms with the same or greater peak capacities. This study shows how to maximize peak capacities using several different columns of different lengths, diameters, and particle sizes. Included in the study is an examination of other factors which affect the measured peak capacities or minimum detectable quantities, such as detector speed, flowcell volume, and mixer volume, for DAD and mobile phase choice, gradient time and flow rate for LC/MS.
P31. Non-Aqueous Reverse
Phase Liquid Chromatography Coupled with Modern Tandem Mass Spectrometry for
Comparative Profiling of Skin Ceramides from ELOVL4
Defective Mice
Johnie
Brown1, Yoshikazu Uchida2 , Vidyullatha Vasireddy4, Radha
Ayyagari4, and Walter M. Holleran2,3
1Applied Biosystems, 500 Old
Connecticut Path, Framingham, MA 01701; 2Department of Dermatology,
School of Medicine, and 3Department of Pharmaceutical Chemistry,
School of Pharmacy, University of California, San Francisco, CA; 4Department
of Ophthalmology, University of Michigan, Ann Arbor, MI.
Corresponding Author Email Address:
krishnsc@appliedbiosystems.com
Epidermal
ceramides (Cer) and their glucosylated counterparts consist of heterogeneous
molecular species, which include different sphingol headgroups (i.e., sphingosine, sphinganine, 4- and 6-hydroxy sphingosine,
and phytosphingosine) combined with amide-linked
fatty acids containing non-hydroxy-, alpha-hydroxy- or omega-hydroxy-fatty
acids. The epidermal-unique omega-O-acylCer species
are formed by the addition of another fatty acid, primarily linoleic
acid, to the omega-hydroxy group. This Cer molecular
heterogeneity is critical for epidermal permeability barrier function required
for mammalian survival. Given that alterations of Cer
molecular profiles are evident in numerous cutaneous
diseases (including atopic dermatitis, psoriasis, and
at least two forms of ichthyoses), a facile,
accurate, and reproducible
method for assessing the structural complexity of epidermal Cer species would represent a powerful tool for cutaneous research. Here we describe a liquid
chromatography (HPLC)-tandem mass spectrometry assay system in which a
non-aqueous reverse phase gradient allows for high chromatographic resolution
over a wide range of lipid types within a single sample injection. A single injection onto the C18 column allows
determination of signal levels from hundreds of individual intact lipid
molecular species from an extract.
Statistical analysis, comparing data from a number of extracts, is then
used to reveal between-sample alterations of individual lipid levels. We recently determined that homozygous mice
deficient in the fatty acid elongation enzyme (Elovl4del/del) have severely
compromised epidermal permeability barrier function. This novel HPLC-tandem
mass spectrometry technique not only revealed the complete loss of epidermal
omega-O-acylCer species in this murine
model, but also yielded an extensive profile of epidermal amide-linked fatty
acid and sphingol species.