product review
Integrated proteomics systems bring the pieces together Several companies offer complete proteomics solutions that include most of the necessary instrumentation. Katie Cottingham
R
esearchers are turning to proteomics in droves, now that genomics isn’t the panacea that it was hyped to be. Experts say that studying proteins is the next natural step because much of the cell’s work is accomplished through protein interactions, which are often controlled by factors that cannot be predicted from the genome sequence. “Genomics isn’t going to help you when it’s not one gene, one protein,” says Bob Bondaryk of Proteome Systems. The splicing of an mRNA transcript can yield tens of different protein isoforms from one DNA sequence. Posttranslational modifications, such as phosphorylation, acetylation, and ubiquitination, can control which proteins interact with each other, where they reside within a cell, and when they are degraded. New tools and techniques are allowing researchers to study an increasing number of proteins simultaneously. Protein chemists are not the only ones excited by these advances—biologists and clinicians are also buying instruments for proteomics projects. Many biologists are using proteomics methods to elucidate vast networks of interacting proteins, while clinical researchers are identifying specific protein patterns, or biomarkers, that may indicate whether a person has a particular disease. Individual components for proteomics studies are available, but over the past few years companies have introduced integrated proteomics systems that bring together all, or most, of the necessary instrumentation into a unified workflow. Table 1, which is meant to be representative rather than comprehensive, lists several integrated proteomics systems, or solutions, that are currently on the market.
Why choose an integrated system? Proteomics systems offer several benefits over stringing together columns, robots, and mass spectrometers on one’s own, say company representatives. Those who will benefit the most from an integrated system are users who “want to be ensured that it’s going to work the first time,” says John Michnowicz of Agilent Technologies. Another advantage is that one company supports the entire system, he adds. Users don’t have to track down support staff from several different vendors if something goes wrong with the instrumentation. Tina Settineri of Applied Biosystems © 2004 AMERICAN CHEMICAL SOCIETY
explains, “The customer will benefit from the company having worked out all the kinks in the process of putting that whole system together.” Although proteomics scientists in academia and industry acknowledge these advantages, not everyone is convinced. “In theory, it’s a great idea, provided that you’ve got really good components,” says John Yates of the Scripps Research Institute. He says a major problem is that companies sometimes sell a mix of good and weak instruments linked together as a system. Other researchers in the field, such as Daniel Figeys at MDS Proteomics, also cite the ability to choose the very best model of each component as the reason they purchase parts separately. Kelvin Lee of Cornell University, however, sees a niche for unified solutions. “I think the kinds of scientists who are making these investments in such integrated solutions are generally people who don’t worry as much about the technology,” he explains. “They just want the answer.” Scientists who study proteins in simple mixtures may not need expensive systems, according to some representatives. Instead of a pre-packaged multidimensional LC/MS system, a single LC column may be sufficient for separating a non-complex sample mixture. Experts also say that integrated proteomics systems often have more complicated plumbing than those put together from individual parts. Such systems can be very difficult to troubleshoot and repair if something goes wrong. S E P T E M B E R 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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product review
Table 1. Selected integrated proteomics systems.1 Product
Nanoflow Proteomics XCT Plus Solution
4000 Q TRAP LC/MS/MS System
4700 Proteomics Discovery System
ClinProt System
Company
Agilent Technologies 5301 Stevens Creek Blvd. Santa Clara, CA 95052 www.agilent.com
Applied Biosystems 850 Lincoln Centre Dr. Foster City, CA 94404 www.appliedbiosystems.com
Applied Biosystems 850 Lincoln Centre Dr. Foster City, CA 94404 www.appliedbiosystems.com
Bruker Daltonics 40 Manning Rd. Billerica, MA 01821 www.bdal.com
Price (U.S.D.)
$250,000–350,000
$445,000
Starting at $395,000
$275,000–655,000
Digestion/sample preparation options
Immunodepletion columns to remove high-abundance proteins; kit for alkylation, reduction, and digestion of samples; lysine tagging reagent for differential expression studies
Not included
Sample plate format compatible with most available digestion workstations; Probot Microfraction collector available
Optional automation package consists of a sample preparation system that loads samples onto patented AnchorChip targets
1-D and 2-D LC
1-D and 2-D LC; accommodates Magnetic bead surfaces capture proteins with certain properties 2-D gels (various surfaces are available)
Separation/fractionation 1-D and 2-D LC, CE; accommodates 2-D gels element
Triple quadrupole/linear ion trap MALDI-TOF/TOF mass mass analyzer with various ion- spectrometer ization sources
MALDI-TOF or MALDI-TOF/TOF mass spectrometer
Data analysis and project Spectrum Mill Proteomics Workbench provides autovalidation of management software MS/MS spectra; open platform for processing data from most MS/MS instruments
Pro ID, Pro ICAT, and ProQuant software with Interrogator database search engine for PTM analysis, protein identification and quantification; Analyst Software for Acquisition and BioAnalyst; LIMS solution available
Protein identification and quantification for gel-based and LC/ MALDI experiments using GPS Explorer software with Mascot database search engine for automated result-dependent analysis; LIMS solution available
ClinProTools software for clinical proteomics and biomarker discovery allows users to visualize large datasets using multiple visualization tools; the software also can cluster sample sets; models are scored based on specificity and sensitivity criteria
Includes methods to identify many proteins with high confidence by focusing on entire sample workflow, especially sample preparation and the processing of data from complex sample sets; mass spectrometer has high-attomole detection levels; optional MALDI source
Automated single-run workflow for PTM analysis, uniquely combining triple quadrupole functionality with linear ion trap capabilities; high-sensitivity absolute protein/peptide biomarker quantitation using triple quadrupole
MS and MS/MS on an easy-touse MALDI platform; complete support for gel-based experiments; complete LC/MALDI system that takes advantage of the off-line nature of MALDI; result-dependent analysis; designed for >50 MS/MS spectra from each well
Enables researchers to prepare and analyze large sample sets with high resolution, high sensitivity, and intuitive software; with TOF/TOF mode, users can also perform higher level analyses, including the identification of biomarkers; captured proteins and peptides can be analyzed on different types of mass spectrometers
Detection element
Special features
Multipole, non-resonance ion trap mass spectrometer
LIMS: laboratory information management system; PTM: posttranslational modification 1 Some companies offer multiple instruments. Contact the vendors for their full product lines.
Because researchers have different needs, most systems are also available in modules, or as individual parts. If a researcher has developed a special LC column, for example, then he or she can often buy the integrated solution without a column. “Complete solutions without a doubt require a large budgetary commitment, which is another reason companies offer modules that can fit with existing instrumentation in customers’ labs or core facilities,” says Bondaryk. He adds that quoted costs often do not include the price of all the necessities. Most integrated proteomics systems include two core elements: a separation/fractionation element and a detection element. Protein separation is typically accomplished by using LC or 2-D gel electrophoresis. A mass spectrometer is included as the detector. Software, both for tracking samples as they progress through the system and for bioinformatics analysis, is generally part of the system. Fraction collectors, robotics for 332 A
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2-D gel spot excision and analysis, and consumables are often either included or offered as options.
Separation and fractionation Proteomics researchers typically separate peptide or protein mixtures in either a liquid-based or a gel-based workflow. Until a few years ago, nearly every proteomics scientist used 2-D gels, which separate proteins by isoelectric point in one dimension and by molecular weight in the second dimension. But extracting proteins from 2-D gels can be time-consuming, and researchers find it difficult to detect membrane-associated proteins and proteins present in low abundance with this method. Multidimensional LC, however, is more amenable to automation, and according to the experts, allows the detection of many proteins that previously were not visible on 2-D gels. Although LC has become popular in recent years, many sci-
product review
Table 1. Selected integrated proteomics systems (continued).1 Product
Proteineer Proteomics Suite
ProteinChip System Series 4000 ProteomIQ
Finnigan ProteomeX LTQ
Company
Bruker Daltonics Fahrenheitstr. 4 D-28359 Bremen Germany www.bdal.de
Ciphergen Biosystems 6611 Dumbarton Cir. Fremont, CA 94555 www.ciphergen.com
Proteome Systems, Inc. 14 Gill St. Woburn, MA 01801 www.proteomesystems.com
Thermo Electron Corp. 355 River Oaks Pkwy. San Jose, CA 95134-1991 www.thermo.com/proteomics
Price (U.S.D.)
$200,000–700,000
$125,000–339,000
INA
$400,000
Digestion/sample preparation options
Options include automated spot Not included detection, spot-picking, digestion, and sample preparation platforms; digestion kits for gel spots; fraction collector
Sample preparation kits; Xcise robotic system for gel imaging, spot cutting, protein digestion, and purification
Not included
Separation/fractionation element
1-D and 2-D LC or surface plas- ProteinChip arrays; chromatomon resonance; accommographic bead chemistry dates 2-D gels and CE
Complete 2-D gel systems and supplies; multi-compartment electrolyzer, a solution-phase IEF device for fractionation prior to 2-D gels or LC
1-D and 2-D LC with four customized, color-coded kits to facilitate the plumbing and automation of specific applications
Detection element
ESI-ion trap MS or MALDITOF/TOF mass spectrometer
MALDI-TOF or ion trap mass spectrometer
Segmented linear ion trap mass spectrometer with hyperbolic rods and radial ejection to dual detectors
2-D gel image analysis; Glycosuite tools for identification of sugar structures on glycoproteins; BioinformatIQ LIMS solution with tools that provide sample tracking, image and spectral analysis, and protein and PTM identifications within a Web-based environment
Bioworks software bundle, which includes the SEQUEST algorithm for confident protein identification, Biomass Deconvolution, DeNovoX for automated de novo sequencing, and SALSA for spectral pattern recognition
Fully integrated platform for collaborative proteomics research, which manages all sample tracking, preparation, protocols, data, and analyses for protein identifications, differential display, and PTM analysis for clinical biomarker discovery
Vacuum MALDI option; ProteomeX LTQ System uses a Spark autosampler designed for low flow rates; can be upgraded with FTICR detector
SELDI-TOF mass spectrometer
Data analysis and project ProteinScape project database Biomarker Patterns software with seamless integration of mul- and Ciphergen Express Biomanagement software tiple search engines; BioTools marker Analysis package and RapiDeNovo for in-depth protein analysis, de novo sequencing, and homology searching; WARP and WARPLC allow MS/MS acquisition control across instrument platforms (ESI/MALDI) Special features
Gel-based workflow is integrated from image analysis to proteomics database; integrated LC/MALDI-MS/MS workflow; AnchorChip target for high-performance MALDI preparation; can sequence intact proteins; can link to ClinProt System
ProteinChip array, software, and kit products for specific applications, such as Expression Difference Mapping, biomarker purification and identification, and Interaction Discovery Mapping for quantitative biomarker assays
ICR: ion cyclotron resonance; IEF: isoelectric focusing; INA: information not available; PTM: posttranslational modification; SELDI: surface-enhanced laser desorption/ionization 1 Some companies offer multiple instruments. Contact the vendors for their full product lines.
entists believe that there will always be a demand for gel-based technologies. “I don’t believe that 2-D gels will be replaced,” says György Marko-Varga of AstraZeneca (Sweden). “They are simply too informative [regarding] intact protein information,” he says. Researchers say that data collected from 2-D gels and LC experiments are complementary and that there’s still plenty of room for both techniques. Detlev Suckau of Bruker Daltonics (Germany) says, “If you want to do a catalog of a proteome, then LC-based workflows can be used. If you’re interested in more details about the proteins, then the gel can tell you such answers.” LC and 2-D gels are not the only options for fractionating and separating proteins. Some systems, such as the Nanoflow Proteomics XCT Plus Solution from Agilent Technologies and the Proteineer Proteomics Suite from Bruker Daltonics, can ac-
commodate CE instruments. The Proteineer system may be combined with a surface plasmon resonance device to screen for protein interactions (for information on other products to detect protein interactions, see Anal. Chem. 2004, 76, 137 A–142 A). The ProteomIQ from Proteome Systems is sold with a multicompartment electrolyzer, which prefractionates complex samples prior to a 2-D gel or LC separation. The electrolyzer “is a compartmented device where the proteins migrate along a pH continuum, moving from one compartment to the next through membranes,” explains Bondaryk. The proteins migrate until they reach a compartment where their charges are neutralized. The device is set up for a narrow pH range so that one can find low-abundance proteins in a complex mixture, he says. Bruker Daltonics’ ClinProt System and Ciphergen Biosystems’ ProteinChip System use chromatographic surfaces to S E P T E M B E R 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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product review
Table 1. Selected integrated proteomics systems (continued).1 Product
MDLC LTQ
Protein Expression System
Protein Identification System
Company
GE Healthcare and Thermo Electron collaboration www.thermo.com/proteomics www.mdlc.com
Waters Corp. 34 Maple St. Milford, MA 01757 www.waters.com
Waters Corp. 34 Maple St. Milford, MA 01757 www.waters.com
Price (U.S.D.)
$450,000
$650,000
$220,000
Digestion/sample preparation options
Not included, but Ettan Spot Handling Workstation, Ettan Spot Picker, and Ettan Digester are available
Optional MassPREP Station for digestion of proteins and preparation for MS analysis; automated protocols
Optional MassPREP Station for digestion of proteins and preparation for MS analysis; automated protocols
Separation/fractionation element
2-D LC Multidimensional LC (MDLC)
UltraPerformance LC (UPLC)
Accommodates LC, UPLC, and 2-D gels
Detection element
Segmented, hyperbolic quadrupole linear ion trap mass spectrometer
QTOF Premier mass spectrometer
MALDI-TOF micro-MX mass spectrometer
Protein Expression Informatics for protein identification and relative quantitation; Expression Informatics analyzes multiple datasets for identification and/or screening of biomarkers or drug targets
ProteinLynx Global SERVER bioinformatics enables project and sample management with routines for protein identification, and imports sample lists, tracks samples throughout analysis and annotates imported 2-D gel images
UPLC has direct nanoflow rate control for improved robustness, reproducibility, and simplicity; proprietary MSE acquisition enables multiplexed precursor/fragment ion analyses; QTOF Premier features programmable Dynamic Range Enhancement, Enhanced Duty Cycle function with T-WAVE technology for PTM detection
Proprietary MassPREP PROtarget and ZOOMtarget plates enable on-target sample cleanup and/or concentration for routine sensitivity at attomolar concentrations; proprietary parallel PSD feature provides extra specificity; data acquisition and analysis support for LC/MALDI
Data analysis and project Unicorn chromatography software platform and/or complete integration with management software Xcalibur MS software
Special features
MDLC includes UV/conductivity detectors, LC flow paths for nano or higher LC flow rates, inert sample path; optional fraction collector; LTQ has excellent ion statistics for fast acquisition speed, MSn sensitivity, spectral quality, true data-dependent analysis, automated MS3 based on neutral loss
PSD: post-source decay; PTM: posttranslational modification 1 Some companies offer multiple instruments. Contact the vendors for their full product lines.
simplify samples before they are analyzed by MS. Porous magnetic beads with reversed-phase, metal-binding, weak cationexchanging, and weak anion-exchanging surfaces are available. Magnets underneath the automation platform move the beads to the side for easy removal of non-binding molecules and liquid. In the ProteinChip System, the chromatographic surface is on a MALDI-type target.
Detection Integrated proteomics systems are currently sold with some type of ion trap or TOF mass spectrometer. “An ion trap gives you MSn capabilities,” says Glen Gregory of Thermo Electron. “Even just going to MS3 opens up a whole new realm of analysis for definite and precise identification of posttranslational modifications,” he adds. There are two main types of ion trap devices: 3-D and linear. Linear ion traps are relatively new on the market, and both Applied Biosystems and Thermo Electron offer these instruments. “Traditional 3-D traps have a limited space inside the trap that you can use, which holds anywhere from 500 to 700 ions or charges. The LTQ [linear trap] can hold 20,000 ions or charges and, more importantly, efficiently use them,” says Gregory. Other companies, such as Agilent and Bruker, have chosen to stay the course and continue developing 3-D models. Michnowicz says, “We feel there’s still a lot more ca334 A
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pacity and capabilities that can be optimized with a 3-D system.” He adds that the Agilent multipole, non-resonance ion trap, for example, can trap 11,000 ions. Applied Biosystems’ linear trap is also a triple quadrupole mass spectrometer, and it can be used as either or as both within the same experiment. Settineri says that this flexibility is particularly useful for the analysis of posttranslationally modified species, such as phosphopeptides. “Phosphorylation always gives a certain m/z or mass fragment,” she says. “The quadrupole is good at looking for just that fragment.” The linear ion trap provides MS/MS data for peptide identification. Although MSn experiments are possible with ion traps, experts say that TOF instruments provide more accurate masses. A compromise may be found in new TOF/TOF instruments, such as those sold by Applied Biosystems and Bruker Daltonics, which representatives say provide both capabilities. Because fragmentation can be induced in a gas cell in a collision energy mode, the quadrupole TOF (QTOF) instrument in the Waters Protein Expression System also provides MS/MS data. This particular QTOF does not require selection of a single precursor ion for fragmentation—in the proprietary MSE mode, all precursor ions are simultaneously fragmented, detected, and quantified. The TOF included in the Waters Protein Identification System features a proprietary parallel post-source decay mechanism that similarly provides MS/MS spectra from
product review
The future
their product lines to meet that demand. For example, Agilent plans to build an integrated solution around its ESI-TOF instrument in addition to the ion trap system already on the market. Also on the agenda for Agilent is the development of new chemistries to facilitate protein expression studies and analysis of posttranslational modifications. Representatives from Bruker Daltonics say that they will introduce magnetic ClinProt beads that researchers can modify with their favorite antibody, antibody fragment, peptide, DNA, or ligand. Researchers say that more work is necessary in the areas of bioinformatics software and in MS performance. Improvements in analysis software could make scientists better equipped to deal with the massive amounts of data that are generated in proteomics experiments. Others add that increases in MS speed, sensitivity, and throughput would be a boon to the field. Whether researchers will latch onto integrated systems is not certain, but proteomics is here to stay, say experts. “I think because of the significance of this information to all sorts of steps in drug development, disease discovery, manufacturing, and protein therapeutics that proteomics technologies will be around for a long time,” says Michnowicz.
Most experts predict an increased demand for integrated proteomics systems, and many companies are planning to expand
Katie Cottingham is an associate editor of Analytical Chemistry.
multiple peptides simultaneously. Electrospray ionization (ESI) and MALDI are popular in proteomics studies. ESI is a liquid-based mechanism that is usually coupled to ion trap instruments. Traditionally, LC fractions are ionized by ESI in real time, but some companies now offer fraction collectors, which give researchers the option to spot the samples onto MALDI plates and analyze them off-line. In the MALDI process, sample droplets are mixed with matrix and dried on a target plate. Although MALDI is typically used with TOF devices, it is becoming more common for companies to offer MALDI sources for ion trap instruments, too. Because ESI and MALDI ionize proteins and peptides via different mechanisms, researchers say that these processes do not always result in the same identifications. “The coverage maps sort of overlap, but a large number of peptides are still quite unique to either technique,” says Suckau. He says that ESI and MALDI are complementary and that Bruker Daltonics researchers run both types of experiments in an integrated fashion and combine the results for database searches.
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