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May 15, 2006 (Vol. 26, No. 10)

Sample Prep Upstream of Mass Spectrometry

Advances Focus on Sample Clean-Up, Fractionation, and Separation

  • Continuing advances in MS technology are both driving and aiding progress in the development of novel sample-prep methods. Advances in MS instrumentation are moving toward higher sensitivity, resolution, and throughput, says John Leite, Ph.D., mass spectrometry product manager at Invitrogen(www.invitrogen.com). These advances, however, have not relieved the burden of sample preparation.

    The ability to advance the capabilities and sensitivity of MS is plateauing somewhat, says John Gebler, Ph.D., director of life sciences at Waters (www.waters.com). New MS techniques and capabilities will continue to emerge; however, for proteomics the emphasis is shifting to the importance of sample prep for improving the reproducibility and robustness of protein sample analysis.

    We would like to demystify the art of proteomics, as it moves toward becoming more of an industrialized science, says Dr. Gebler. This would involve adopting more standards, quality control, and validated methods, similar to what exists for the identification of small molecules in biological fluids.

    One of the most significant trends in sample-prep technologies has been their application to biomarker discovery, according to Julie Hey, senior product manager in the lab separations division at Bio-Rad Laboratories (www.bio-rad.com). This is especially apparent in the plasma/serum proteomes because of the potential for clinical applications and also the ease in which plasma/serum can be obtained.

    Regardless of whether 2-D gel electrophoresis or LC technology, including multidimensional liquid chromatography (MDLC) techniques, is used to separate complex protein mixtures, there will be continued demand for tools and technologies to reduce sample complexity and enrich for low-abundance proteins, Hey says.

    The advancing power and sensitivity of mass spectrometers will continue to dictate how much sample prep is necessary. The key is fractionation, says Hey. The better the separation prior to the mass spectrometer, the higher the success rate in getting high-confidence protein identification.

    In addition to an emphasis on increased automation and standardization, proteomics research is moving toward miniaturization technologies to improve throughput, minimize resource utilization, and decrease costs.

    In the future, even further miniaturization will be possible by using chip platforms for protein and peptide sample preparation and separations, predicts Remco van Soest, product manager of nano LC at Eksigent Technologies (www.eksigent.com). Devices like these ideally would be for one-time-only use to reduce any possibility of carryover.

    An example of this trend is Agilent Technologies’ (www.agilent.com) 1200 Series HPLC-Chip/MS system, a microfluidic chip-based technology designed for nanospray LC/MS. All components of the system, including sample prep and separation columns, connection capillaries, fittings, and nanospray tip are integrated on the re-useable polymer HPLC-Chip.

    New to the market are Qiagen’s (www.qiagen.com) Mass.Spec Chips, which enable on-target sample processing. Mass.Spec.Focus Chips perform on-chip concentration and clean-up of proteomics samples. Each chip contains virtual wells that comprise a central target area and are surrounded by a hydrophobic non-wettable surface to prevent cross-contamination.

    Samples (up to 35 µL) are spotted onto the chip and allowed to evaporate. A drop of matrix solution is then added to each well. It is allowed to dry and cofocus with the resuspended sample. Desalting chips contain a functionalized surface that binds peptides, and salts are removed through a wash step. IMAC chips use immobilized metal ions for purifying metal-chelating peptides, such as phosphopeptides, from protein digests.

    Qiagen designed its Mass.Spec.Turbo chips for high-throughput MALDI MS peptide analysis. Users pipette samples onto MALDI matrix spots either manually or using a microfraction collector; a vacuum process for spot deposition in manufacturing produces homogeneous crystals.

  • Biomarker Discovery

    Several key factors are driving technology and product development for sample prep in proteomics-based research and protein analysis. These include an emphasis on biomarker discovery and the search for clinically relevant proteins, which are likely to be present in tissues and body fluids in relatively small quantities. This will require continued improvements in the robustness and reproducibility of separation techniques upstream of MS and innovative methods for fractionating the proteins in biological samples and targeting protein families of interest.

    The key to successful biomarker discovery, identification, and protein characterization is effective sample prep, says Jerry Feitelson, Ph.D., strategic product manager at Beckman Coulter (www.beckmancoulter.com). According to Dr. Feitelson, partitioning of the highly abundant components of the proteome and subsequent fractionation of the moderate to low abundance proteins are critical steps in preparing samples for MS analysis.

    In these highly complex samples, without simplification and enrichment of target proteins it is virtually impossible to dig deep enough into a proteome to characterize biologically important differences, Dr. Feitelson says.

    Together, proteome partitioning and fractionation have the combined effect of enriching for low-abundant proteins and removing the masking effect created by complex mixtures. Our customers have demonstrated that combining these two approaches permits identification of low-abundance serum proteins in the sub-nanogram/mL range, such as Troponin I, adds Dr. Feitelson, Protein identification at this depth is a breakthrough in this industry, paving the way for the discovery of many relevant biomarkers of high importance.

    Proteomics researchers often want to focus on a single cell type or sub-structure, and approaches such as laser micro dissection and cell sorting by tagging cell surfaces represent promising techniques for looking more closely at a specific set of proteins. These approaches, however, increase the need for high sensitivity in all aspects of sample preparation and analysis, says Anke Cassing, Ph.D., associate global business director for proteins at Qiagen.

    In MS sample prep, we envision an increasing demand for enrichment of subsets of peptides, Dr. Cassing adds, including peptides carrying post-translational modifications, automation of sample prep processes, tagging strategies to create affinity tags for peptide subsets, endo-proteases or other chemical methods for cleaving target proteins at specific position, and commercially available micro-chromatography tools suitable for MS-scale applications.

    Another beneficial sample-prep strategy would enable MS analysis directly from fractionated proteins (gel-free protein pattern profiling), says Dr. Cassing.

    As mass spectrometers are becoming more sensitive and generating more data, researchers are looking to focus their experiments more narrowly by fractionating the proteome upstream of the protein separation step and targeting one specific portion of the proteome, according to Jasmine Gruia-Gray, marketing director of protein discovery at GE Healthcare (www.gehealthcare.com).

    A new technology in development will enable fractionation to be done downstream of sample separation and upstream of MS analysis. GE Healthcare is developing a microfractionation and spotting device that would collect fractions coming off the HPLC and spot individual fractions onto MALDI targets.

    Greater ease of use and increased reliability of MS instrumentation has steadily been increasing access of MS technology to biochemists and biologists who are closer to the sample-prep side of the proteomics workflow, says Jerome Bailey, program manager for bioseparations at Agilent. Advances in instrument scan speed coupled with HPLC column technology (microfluidics) are improving overall ease of use, enabling a deeper and more complete measurement of the proteome, and improving sample throughput.

    To satisfy customers’ desire for ease of use, reproducibility, and throughput in www.bruker.com) developed the Prespotted AnchorChip (PAC) targets dedicated to proteomics applications. The targets comprise homogeneous, thin layers of HCAA (alpha-cyano-4-hydroxy-cinnamic acid) matrix and peptide standards to simplify calibration. The targets enable researchers to archive protein samples for several months in amounts down to the subfemtomolar range. The PAC targets, available with 96- or 384-sample spots, require a maximum two-step sample preparation, including in situ sample purification.

    The field is moving away from classic MUDPIT (multidimensional protein identification technology)which is a good tool for survey workto multistage separation techniques, says Roger Biringer, senior scientist in proteomics R&D at Thermo Electron (www.thermo.com). The more separation steps in the process, the more likely it will reveal low-abundant proteins, he adds. One commonly used multistage separation method involves SCX pre-fractionation of proteolytic digests followed by reverse-phase LC/MS of the individual fractions.

    Now that detectors are available that can handle larger peptides, and even proteins, Biringer reports a growing interest in doing reverse-phase separations of larger peptides using some of the newer monolithic technologies, which offer fast and fine separations. He also reports a trend toward incorporating a chromatographic protein step before protein digestion, eliminating the need for 2-D gel electrophoresis. This could involve an initial separation step using a monolithic solid-phase extraction (SPE) media, followed by protein digestion, and then ion exchange chromatography and reverse-phase LC/MS.

  • Sample Clean-up

    Clean-up upfront of the mass spectrometer is essential, as the cost of getting the mass spec dirty inside is prohibitive, says John Siira, product manager at Caliper Life Sciences (www.caliperls.com).

    Furthermore, detergents and ion suppressing salts can compromise MS analysis. Sample clean-up is also an important issue prior to 2-D gel electrophoresis and LC to ensure robust and reproducible results. For example, salts can interfere with isoelectric focusing in the first dimension of 2-D gel electrophoresis.

    Upstream of MALDI MS and LC/MS it is necessary to remove the detergents, salts, and chaotropes commonly used in proteomic sample preparation to increase protein solubility. Too often, though, methods to remove these contaminants force scientists to compromise protein yields for high-quality data, says Invitrogen’s Dr. Leite.

    Since many pharmacologically valuable targets are membrane proteins, high sequence coverage is required in order to accurately map sites of post-translational modification and sites of agonist/antagonist binding, elucidate stoichiometry, and identify sites of protein-protein interaction. Due to the amphipathic nature of these hydrophobic proteins, the hydrophilic regions are preferably detectable over hydrophobic regions, resulting in poor sequence coverage. Thus, reagents that can increase sequence coverage of hydrophobic proteins are in great demand, says Dr. Leite.

    Advantages of Invitrogen’s Invitrosol MALDI Protein Solubilizer include the ability to prepare both hydrophobic and hydrophilic proteins or peptides directly for MALDI-TOF MS analysis without the need for subsequent acid hydrolysis, SPE, or crystal washing, thereby minimizing sample loss. Similarly, direct injection of Invitrosol LC-MS into a reverse-phase column is possible without the need for a separate ion exchange step or column washing.

    High-throughput processes may not be necessary for all applications, says Travis La Favor, market segment manager for sample prep at Pierce Biotechnology (www.piercenet.com). Existing technologies for proteomic sample prep meet a range of research needs.

    Pierce offers multiple options for sample clean-up, including spin columns prefilled with C18 media for MS clean-up, desalting columns, and high-throughput Zeba 96-well desalting spin plates.

    The company’s 2D Sample Prep Kits for soluble and insoluble proteins remove salts, buffers, and other small ionic contaminants from cultured cell extracts and tissues upstream of 2-D gel electrophoresis and MS. Pierce also recently introduced the 2000 MWCO Slide-A-Lyzer dialysis cassettes.

  • Protein Sample Depletion

    During the past three to four years, the strong market for 2-D gel electrophoresis has led to the development of tools that streamline the workflow upstream of 2-D gel electrophoresis and MS, and simplify the proteome, notes Karin Hughes, director of product management for pathway analysis at EMD Biosciences (www.emdbiosciences.com). EMD, through its Novagen and Calbiochem brands, introduced a line of mutually compatible ProteoExtract and ProteoEnrich kits to address this need.

    Tsetska Takova, product manager for protein sample prep and purification at EMD, describes how experiments that search for rare biomarkers might first involve depletion of high-abundant proteins, such as albumin or albumin/IgG, followed by the use of the ProteoEnrich CAT-X and CAT-X-SEC kits to further reduce proteome complexity under nondenaturing conditions based on binding to the strong cation exchange resin Fractogel EMD SO3 or to a unique resin, respectively, that allow for partial proteomes and low molecular weight protein enrichment by eluting with a salt gradient.

    The next step in the sample-prep workflow would depend on several factors, including the downstream separation process and the associated need for dialysis to remove salts, for protein precipitation, for the removal of other contaminants, and for protein digestion. These dialysis, digestion, and other steps can be combined with additional rounds of sample enrichment to maximize the quality and purity of the sample that will ultimately be evaluated by MS analysis.

    Agilent’s Bailey brings into focus the issues surrounding depletion of high-abundance proteins: There is still the open question of how many proteins is it optimal to deplete? Tightly coupled with this question is what product performance is needed to realize maximal value from depletion?

    Enabling depletion of 99% of 20 high-abundant human plasma proteins, Sigma-Aldrich’s (www.sigmaaldrich.com) ProteoPrep 20 Plasma Immunodepletion kit is designed for front-end fractionation upstream of 2-D gel electrophoresis or HPLC. The kit contains multivalent antibody affinity media in a spin column format. It removes 97% of the overall protein mass in human plasma, according to Dale Peluso, market segment manager for quantitative proteomics.

    There is a market building to look for biomarkers in other biofluids, such as saliva, urine, and cerebrospinal fluid, says Peluso, and Sigma-Aldrich is expanding its protein fractionation and depletion technology for use in additional types of biological samples.

    In April, Agilent introduced a high-capacity column that removes the seven most abundant proteins in human plasma. The company re-engineered its Multiple Affinity Removal System to double the standard sample-loading capacity (µg of protein/mL of resin). The system utilizes polyclonal antibodies affinity purified with native human plasma antigens to ensure low species cross-reactivity and employs buffers formulated to minimize protein-protein interactions.

    Beckman Coulter’s ProteomeLab PF 2-D LC system is compatible with the company’s low-abundant protein enrichment chemistries. The ProteomeLab IgY-12 spin and LC column kits selectively partition 12 highly abundant proteins found in serum or plasma. Multiple formats allow for sample processing of 20 to 250 microliters per cycle. The IgY chemistry has been optimized for both human/primate and rodent samples; however, a key advantage is its ability to partition orthologous proteins across species, according to the company, allowing animal models and human samples to be processed using the same chemistry.

  • Protein Enrichment and Extraction

    As innovations in sample prep move toward miniaturized formats, faster processes, and cleaner samples, the focus will continue to intensify on developing better techniques for fractionating samples and identifying rare proteins.

    We need to go further, says La Favor, to develop fractionation methods that enable the isolation and purification of low-abundance proteins. For protein fractionation, Pierce offers its 2-D Sample Prep and Poppers kits capable of enriching for nuclear, cytoplasmic, and mitochondrial proteins, as well as the new Pinpoint Cell Surface Protein Isolation kit, which uses biotinylation to purify mammalian cell surface proteinsimportant for receptor binding studies in drug discovery research.

    For high-abundant protein removal, the company developed the ProteoSeek antibody-based albumin/IgG removal kit and SwellGel albumin removal discs, designed for high capacity (about 2 mg) albumin removal from small (10� µL) serum samples.

    The choice of targeted enrichment strategies will depend on how much is known about the protein or family of proteins of interest. For example, EMD Bioscience’s ProteoExtract Subcellular Proteome Extraction kit enriches for four subcellular compartments, allowing users to isolate, for example, the cytosolic or mitochondrial sample fraction. The company’s ATP-Binders kit enriches for ATP-binding proteins, such as kinases, whereas the ProteoExtract Native Membrane Protein Extraction kit extracts membrane proteins, and the Phosphopeptide Capture kit targets phosphopeptides.

    Waters introduced a set of phosphopeptide standards late last year and is developing methods to use nanoflow LC to enrich and purify phosphopeptides. The company also offers the MassPREP product line, which includes a peptide standard mixture, protein digest standards, MALDI matrices, online desalting cartridges, and a glycoanalysis kit.

    Bio-Rad’s protein sample fractionation products include the ReadyPrep protein extraction kits that fractionate protein mixtures based on cellular location (the cytoplasmic/nuclear fraction, membrane proteins, and membrane proteins specifically involve in intracellular signaling), or based on differential solubility.

    Qiagen’s Qproteome fractionation kits enable isolation of cellular organelles (separating out proteins found in the cytosol, membranes, nucleus, or cytoskeleton), soluble proteins, phosphoproteins, and glycoproteins, and depletion of high-abundant proteins such as IgG and albumin.

    The company’s newest additions to the product line are the Qproteome Plasma Membrane Protein kit, which separates plasma membrane proteins from other membrane proteins, and the Qproteome Mitochondria Isolation kit.

    Proteomics samples should reflect the dynamic range of a proteome, says Cassing. For quantitative applications, the protein mixture should possess at least a dynamic range of 3 (or even 5) orders of magnitude. Furthermore, the sample should be highly reproducible and reflect a real-life situation, covering a range of proteins, including proteins as positive and negative controls that offer challenges such as hydrophobicity, stickiness, Cys-richness, over- or under-representation of tryptic cleavage sites, and trends for oligomerization, for example.

  • Solid-Phase Extraction

    Demand for higher throughput and greater automation capability contributed to Varian’s (www.varian.com) expansion of its OMIX product line for microextraction of proteins and peptides using SPE. In addition to its existing OMIX pipette tips, Varian introduced OMIX 96-well plates and OMIX tips compatible with the Tomtec (www.tomtec.com) Quadra.

    The company’s C18 pipette tips, containing a reverse-phase resin for salt removal, its strong cation exchange (SCX) tips for removing detergents, and its C4 tips for desalting and concentration of larger, intact proteins, all contain monolithic, porous silica-based SPE media that offers improved flow properties and binding capacities compared to packed bed media, according to Jennifer Massi, market development manager for SPE products at Varian.

    Because MALDI MS plates only accommodate about 1-µL sample spots, elution volumes for proteomics need to be small, says Massi. Varian will continue to expand its OMIX product line into additional formats and phases, for example, adding functional groups designed to enrich for phosphorylated proteins.

    The RapidTrace SPE Work Station, Caliper Life Sciences’ automated high-throughput SPE platform, is a syringe barrel-based device designed for protein extraction in biological samples at volumes of 200 µL to several milliliters. Each RapidTrace module can accommodate 10 SPE cartridges and process up to 100 samples in less than two hours.

    The ability to run a different method on each sample in an automated platform makes the RapidTrace useful for methods development work to generate a rugged method, says Lynn Jordan, manager of the application science and technology laboratory at Caliper.

    For higher-throughput SPE in a 96-well format, Caliper’s Sciclone ALH 3000 conditions the 96-well SPE plate, then loads samples from a 96-well plate into the SPE plate, performs the extraction, and elutes the samples into a clean microtiter plate.

    Caliper’s newest entry in the SPE market is the Sciclone i1000, which can dispense to 96- or 384-well plates. Like the RapidTrace, the Sciclone i1000 can perform different experiments at variable volumes in each individual well. To avoid problems with clogging, well overflow, and plate contamination when working with biological samples, the i1000 uses a pipette tip to sense the presence of liquid remaining in a well, and can then turn off the pipettes servicing any clogged wells, continuing to process the rest of the plate, according to the company.

    For desalting and concentration of protein digests derived from 2-D gel electrophoresis experiments in preparation for MS analysis, Millipore (www.millipore.com) developed the ZipTip, a 10-µL pipette tip containing a bed of chromatography media fixed at its end. The ZipTip purifies and concentrates femtomoles to picomoles of protein or peptide with no dead volume and low elution volumes appropriate for MS applications.

    Millipore’s ZipPlate micro-SPE technology enables in-solution or in-gel digestion, which collapses several steps of the process together, minimizes sample loss and transfers the concentrated, digested proteins directly onto a MALDI plate surface, says Mark Kavonian, group product manager for protein research at Millipore. For fractionation applications, ZipTip MC was designed for phosphopeptide capture, and ZipTip SCX contains a strong cation exchanger for detergent removal and fractionation.

    In March, Millipore introduced a new family of solid-phase extraction cartridges containing 3M Empore extraction disk membranes. Available sorbents include C2, C8, C18, universal resin, and mixed-phase cation resin. The Milli-SPE cartridges join the company’s 96-well Multi-SPE Extraction Plates, which are designed for higher-throughput applications.

    Kavonian describes a combined ultrafiltration (UF) and SPE approach to proteomic sample preparation from serum that eliminates the need for both gel electrophoresis and chromatography steps, yielding a sample that can be applied directly for MS or MS/MS analysis. The method involves the fractionation of small proteins or peptides using either an Amicon Ultra 10K centrifugal UF concentrator (low-throughput method) or a Ultracel 10K plate (high-throughput method). The filtrate is transferred to either C18 ZipTips or a 96-well ZipPlate, respectively, for desalting and concentration and is then eluted onto a MALDI target.

  • 2-D Gels & LC/MS

    One of the main trends in proteomics research is the focus on specific classes of proteins that can influence cellular cascades and cross-talk, according to Gruia-Gray.

    This would include G-coupled proteins, but also glycosylated, phosphorylated, and ubiquinated proteins, as well as lipoproteins. The desired class of proteins would guide the selection of the most appropriate separation technology. Proteins at the extremes of pH or that are very hydrophilic or hydrophobic, for example, will not separate as well on a 2-D gel as they would on MDLC. On the other hand, 2-D gel electrophoresis has the advantage of being able to load more protein.

    There are trade-offs, says Gruia-Gray. You can run the exact same sample in a MDLC workflow versus a 2-D workflow and get a percentage of the same answers (proteins identified) and a percentage of totally different answers.

    With the trend toward demand for quantitation of the proteins in a sample, 2-D gel electrophoresis offered a distinct advantage over MDLC. However, the introduction of reagents such as iCAT, iTRAQ, and iPROT brought quantitation capability to MDLC methods.

    Elena Chernokalskaya, director of technology development at Millipore, describes the blending of these two complementary separation techniques. For example, a sample might first be fractionated based on isoelectric point using isoelectric focusing (IEF) fractionation devices. Each fraction could then be separated by 1-D SDS-PAGE, the bands excised, in-gel digestion performed, and the proteins identified by LC/MS/MS. Another example would be the separation of proteins using one or two chromatography steps prior to SDS-PAGE.

    2-D gel electrophoresis/MS and LC/MS are complementary approaches, explains Hey of Bio-Rad. Each has its own advantages and disadvantages, and each identifies a different set of proteins. Ideally, proteomic workflows would include both technologies to maximize the information generated.

    Hey identifies several advantages of 2-D gel electrophoresis: it can visualize post-translational modifications (modified versus unmodified peptides will have different mobility in a 2-D gel); it enables protein quantification; and its initial set-up costs are substantially lower than LC/MS. For ease of automation, though, LC/MS has definite advantages, notes Hey.

    2-D will definitely evolve toward automation, but it will have to be available at the same relative cost as the current manual process, she says. Previous attempts at automation have failed most likely because they were cost-prohibitive.

    Sample prep is an obvious bottleneck in the proteomics workflow, says Dr. Leite. Automation will certainly alleviate issues surrounding cost and throughput.

    GE Healthcare’s Ettan line of hardware, software, and reagents for protein separation and protein-expression analysis includes products for performing nano-scale LC (Ettan MDLC and Ettan nanoLC); 2-D DIGE (direct differential analysis of two or more protein samples on one gel); gel-imaging technology, spot-picking instrumentation; labeling reagents such as CyDye DIGE fluors; new Amersham ECL Plex (enhanced chemiluminescence) labeling reagents for quantitative fluorescent Western blots from 1-D gels; Immobiline DryStrip gels for isoelectric focusing to fractionate protein samples; and DeStreak reagent to remove contaminants in the first dimension of 2-D gel electrophoresis.

    Bio-Rad offers a full spectrum of 2-D gel electrophoresis products, from sample prep through to spot cutting, imaging technologies, and image-analysis software. Recently introduced products include the MicroRotofor isoelectric focusing cell; a miniature preparative IEF device that fractionates complex protein samples in free solution; Flamingo fluorescent gel stain; Proteomweaver for automated spot detection, quantitation, and matching; and the EXQuest spot cutter.

    Proteomic sample-prep tools vary widely and differ primarily based on the amount of sample they can accommodate, the depth and breadth of proteome coverage achieved, the degree of fractionation and extent of enrichment, and their ease of use, cost to operate, and throughput, according to Dr. Feitelson.

  • Innovation in LC/MS

    Beckman Coulter’s ProteomeLab PF 2D (Proteome Fractionation in 2 Dimensions) System fractionates a proteome in the first dimension by pH using chromatofocusing, and then automatically injects each fraction into the second dimension for separation by hydrophobicity.

    The system generates liquid fractions of the proteome as well as a pH/hydrophobicity 2-D map that identifies regions of differential protein expression. With up to a 5-mg mass load, the system allows the isolation of low-abundant proteins at levels that can readily be identified by mass spectrometry, according to the company.

    Over the past five years, Dominic Gostick, Ph.D., director of proteomics for the ToF-MS product line at Applied Biosystems (www.appliedbiosystems.com), has observed a trend away from traditional 2-D gel electrophoresis and a protein identification-based workflow to an LC/MS workflow and an emphasis on identification and quantitation. The introduction of istopic and isobaric labeling reagents has enabled relative quantitation of proteins and multiplexing at the level of the mass spectrometer.

    The move from a 2-D gel world to the world of LC and mass spectrometry has enabled people to better deal with the complexity in their proteomics samples by having a liquid chromatography step upfront, says Dr. Gostick.

    At the end of 2005, Applied Biosystems introduced Tempo LC, a range of low-flow chromatography systems designed as front-ends for either ESI or MALDI MS. In January, the company launched the QSTAR Elite LC/MS/MS system, designed to offer improved resolution, dynamic range, and speed of MS/MS for proteomics applications. The biggest challenge today is dynamic range, says Dr. Gostick. Both 1-D and multi-dimensional chromatography are enabling us to go deeper into the proteome.

    Eksigent Technologies’ nano-scale LC systems designed for nanospray mass spectrometry incorporate microfluidic pumping technology to deliver precise, low-flow rates down to 100 nL/min gradients, without the need for flow splitting. The Nano LC-1D and 2-D systems integrate with MS instrumentation from major manufacturers through Eksigent’s LC Control software to enable LC/MS identification of proteins and peptides in proteomics applications such as biomarker discovery.

    The 2-D system can perform online multidimensional separations, typically with an ion exchange column in the first dimension and a reverse-phase column in the second dimension. The company introduced the Nano LC-1D Plus in 2005, which includes a third pump for rapid sample loading and washing.

    Bailey identifies five main areas in which HPLC-based sample-prep strategies will evolve. These include new affinity-capture technologies; continued integration of protein separations with LC/MS hardware and software; increased recovery of protein samples during sample prep and separations; continued improvement of multidimensional separation techniques to enable new workflows for biomarker discovery; and greater emphasis on whole protein capture and separations to preserve and maintain post-translational modifications information.

    Bailey emphasizes industry demands for improved reproducibility, increased sample-loading capacity, high protein recovery workflows, better selectivity, reduced sample-preparation time, and increased automation.

    Agilent’s proteomics separation suite consists of four main product families: the multiple affinity removal system, macroporous reverse-phase (mRP) columns, HPLC chip products, and the soon-to-be-released OFFGEL Fractionator.

    GE Healthcare’s Ettan MDLC and Ettan nanoLC can be used in combination with the company’s new Decyder MS differential analysis software, which allows users to import retention times/chromatograms with the mass-to-charge data from the mass spec and analyze the LC/MS data as a 2-D plot or a 3-D intensity plot. In this way you can visualize anomalies in the data that couldn’t as easily be seen on the mass spectra or chromatograms, explains Gruia-Gray. These findings can be used to evaluate the quality and reproducibility of sample-prep procedures.

    You can take DeCyder MS one step further, adds Gruia-Gray, and do the equivalent of DIGE, comparing samples, doing differential analysis, and doing relative abundance comparisons.

    Waters’ nanoACQUITY Ultra Performance LC (UPLC) system performs capillary to nano-scale separations at flow ranges of nL/min up to µL/min in 75? micron diameter columns. The nanoACQUITY system does not require flow splitting, an advantage for customers, notes Gebler, because as the column is aging and they are putting on different kinds of samples, they don’t have to worry about the flow characteristics of the column changing. The nanoACQUITY also offers online MDLC capability.

    Other Waters products with applications in proteomic sample prep include RapiGest SF reagent, BioSuite HPLC columns, Protein-Pak columns, the Sep-Pak line of SPE products, and the NanoEase nano, capillary, and trapping columns.

    Thermo’s newest entry in the LC market is its line of Hypersil Gold columns. These 1.9-micron particle chromatography columns offer constant efficiency over a large flow range, says Biringer. The company’s protein separation and analysis product line includes the Finnigan LTQ, OrbiTrap, and FT hybrid mass spectrometer. In 2006, the company introduced Electron Transfer Dissociation MS as an upgrade for the LTQan ion fragmentation technology that dissociates peptides to generate sequence information.



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