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Quantitative proteomics comes of age Methods for quantitating proteins by MS increase in concentration. Jennifer Griffiths
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n the past decade, the field of proteomics has been playing out its own modern version of the tortoise and the hare fable. Plodding along as steadily as the tortoise was the reliable workhorse of the proteomics field, two-dimensional gel electrophoresis (2DE). Meanwhile, the rabbity upstart technique of MS was rapidly outpacing 2DE and drawing more and more proteomics researchers over to its side. Clearly, MS was faster and better at identifying proteins, but it had its own shortcomings, particularly in the area of quantitative proteomics. To measure protein abundance, most researchers relied on 2DE/MS, a laborious technique whereby proteins are quantitated in-gel and then individually cut out, digested, and analyzed in a mass spectrometer. To win the race, MS needed a fast and reliable method to quantitate proteins. Luckily, the proteomics research community has stepped up to the challenge. “The wonderful thing about quantitative MS proteomics now is that we have so many new tools. If you go back a few years, we essentially had nothing,” says Philip Andrews of the University of Michigan. Most of the techniques use stable isotope ratios to measure protein abundances, and several methods have now matured to the point that their reagents have become commercially available. These products are listed in the tables accompanying this article. Quantitation experiments can be divided into three camps: those that chemically tag proteins or peptides after cell lysis (Table 1); those that tag during cell growth (Table 2); and those in which the sample is spiked with a known amount of an isotopically labeled peptide derived from the target protein for comparison (Table 3). Note that the tables are meant to be representative, not comprehensive; vendors may offer similar products not included here.
Why do we need isotopes?
By all accounts, MS is excellent at identifying what is in a sample. A typical MS proteomics experiment starts with a trypsin digest of the cellular extract. The resulting peptides are fragmented for MS/MS analysis, and their sequences are derived by comparison with a spectral database. The identification of peptides unique to a protein confirms its presence in the sample. © 2007 American Chemical Societ y
Knowing that a protein is present is useful information, but many proteomics researchers also need quantitative data to reach meaningful conclusions about their experiments. For example, researchers looking for a biomarker of a given disease want to compare protein concentrations in healthy versus diseased tissue. The simplest methods for MS quantitation count the number of fragment ions or peptides that a protein produces. These “spectral counting” methods are fraught with errors because peptides of different sequences behave differently in a mass spectrometer and larger proteins produce more peptides. A major difference in ionization efficiency, for example, can dramatically skew abundance ratios, and therefore these methods can give only rough estimates of concentration. For a more accurate picture of protein quantity, researchers have turned to stable isotopes. For these experiments, peak heights of unlabeled or isotopically light peptides are compared with those of isotopically heavy labeled versions of the same sequence. Because the sequences are identical, the peptides ionize and fragment the same in any mass spectrometer. Thus, the relative abundance ratios in the mass chromatogram are directly proportional to relative expression ratios. Two types of quantitative data can be derived from stable isotope experiments: the relative change in a protein’s concentration between two or more samples, and the absolute S e p t e m b e r 1 , 2 0 0 7 / A n a ly t i c a l C h e m i s t r y
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amount of that protein in a sample. Most of the commercially available methods compare abundances among samples. John Asara of Beth Israel Deaconess Medical Center says that “with most of the stuff that we do, it’s much less important
generation version of the ICAT reagent, now available from Applied Biosystems, uses a 13C/12C labeling scheme and includes an acid-labile cleavage site to remove the biotin group after purification. “The benefit of ICAT is that it helps in simplifying the samples because the protocol includes Acronyms for stable isotope labeling methods an affinity purification step,” says Subodh Nimkar of Applied Biosystems, but he also points out AQUA Absolute quantitation that because it labels cysteine, only a relatively ICAT Isotope-coded affinity tag small percentage of the protein population will be ICPL Isotope-coded protein label tagged. Another limitation of ICAT is that only two versions of the tag are offered, restricting it iTRAQ Isobaric tagging for relative and absolute quantitation to pairwise comparisons of samples. SILAC Stable isotope labeling by amino acids in cell culture Many researchers have moved away from ICAT technology in favor of newer methods, though to know the absolute quantity of a peptide, but it’s very imthe technique is still favored for some specific applications. portant to know how that level changes between conditions.” “Our main application for ICAT is when we are looking at From this relative change, investigators can infer a system’s changes in the oxidative state of cells,” says Andrews. “We biological response to a drug or other stimulus. Still, some know that there’s going to be a change in the disulfide bonds, classes of experiment, such as diagnostics, require an absolute so we’ll specifically use the ICAT reagent because it gives us quantitation of one or more markers. Methods and reagents selectivity for the cysteine residues.” for both types of experiments are described below. One of the most popular replacements for ICAT is a method called iTR AQ, also available from Applied BiosysChemical tagging outside the cell tems. The method was developed when the company recogThe largest number of commercially available methods modify nized the need for higher level multiplexing, which ICAT proteins at either the protein or peptide level after cell lysis. couldn’t do. The iTR AQ reagent, which can label at either These methods are the most general measure of relative change the protein or peptide level, consists of three parts: an amineand can be used with proteins from all sources, including cell targeting N-hydroxysuccinimidyl moiety, a reporter group, culture, serum, and tissue extracts. Most of these reagents and a mass balance group. The iTR AQ tag is an isobaric one, target either thiol or amino groups; one method labels the car- which means each of the four versions of the iTR AQ tag has boxy terminus of peptide fragments. the same mass before MS/MS fragmentation; after fragmenIn a typical experiment, two or more samples would be tation, the reporter peaks appear between 113 and 121 Da. subjected to tagging by reagents that are of identical chemical structure but that Table 1. Selected ex vivo tagging reagents.1 differ in the carbon, hydrogen, oxygen, Product Company Application notes or nitrogen isotopes that they contain. Labels thiols; includes acid-cleavable biotin After tagging, the samples are combined ICAT reagents Applied Biosystems and kits 800-327-3002 group for selective purification; quantitated in and processed together. Each fragment www.appliedbiosystems.com MS mode; duplex labeling; labels at protein level ion that contains the isotope label will Applied Biosystems Labels amines; isobaric mass tags quantitated in give multiple peaks, one for each version iTRAQ reagents and 800-327-3002 MS/MS mode; up to 4-plex labeling; company has of the tag; comparison of peak heights kits www.appliedbiosystems.com announced plans for an 8-plex version gives the relative abundance of each 18 O water Cambridge Isotope Laboratories Labels at carboxy terminus during trypsin digesspecies. 800-322-1174 tion; duplex labeling; cheaper than many other The “grandfather technology” of ex www.isotope.com methods vivo tagging is a method called ICAT, ExacTag kits PerkinElmer Labels thiols or amines; isobaric mass tags quanaccording to Peter Banks of PerkinEl800-762-4000 titated in MS/MS mode; 2-, 4-, 7-, or 10-plex kits www.perkinelmer.com available; has dedicated free software that works mer. The tag, which is applied at the with common search engines protein level before proteolytic diges18 tion, was originally described by Ruedi O Proteome Sigma-Aldrich Labels at carboxy terminus during trypsin digesProfiler Kit 800-325-3010 tion; duplex labeling; cheaper than many other Aebersold, now at ETH Zurich, and www.sigmaaldrich.com methods colleagues in 1999 (1). The original ICPL kit Serva Electrophoresis GmbH Labels amines; 3-plex; works the best with Bru tag consisted of a thiol-specific reactive (Germany), distributed in the U.S. ker instruments because of software issues group, a linker that incorporated either by Crescent Chemical Co. hydrogen or deuterium at eight posi631-348-0333 www.crescentchemical.com tions, and a biotin group for selective 1 Some companies may offer similar products not listed here. Contact the vendors for their full product lines. capture of labeled peptides; a second6452
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Table 2. Selected in vivo tagging reagents.1 Product
Company
Application notes
Stable-isotope-labeled amino acids
Cambridge Isotope Laboratories 800-322-1174 www.isotope.com
Wide range of specific and uniformly labeled amino acids available
SILAC protein ID and Invitrogen quantitation kits 800-955-6288 www.invitrogen.com
SILAC protein quan- Pierce Biotechnology titation kits 800-874-3723 www.piercenet.com
Labels during cell culture; kits available to target phosphoproteins and membrane proteins; kits contain 13 C-labeled Lys; 13 C-, 15 Nlabeled Arg sold separately Labels during cell culture; kits contain 13 C-labeled Lys; 13 C-, 15 N-labeled Arg sold separately
1
Some companies may offer similar products not listed here. Contact the vendors for their full product lines.
When compared with ICAT, which is analyzed in MS mode, “the iTR AQ-based quantitation is more sensitive and more selective because there is no background in the MS/MS mode,” points out Nimkar. In addition to the more mature technologies, two newer tags have recently been introduced to the market. Serva offers an ICPL reagent that operates on the same principles as ICAT but labels amine groups rather than thiols, resulting in much higher sequence coverage (2). Thomas Halder of Toplab (Germany) says a big advantage of ICPL is that it labels at the protein level and is very specific. “If you keep the pH like it is described in the manual, you won’t see any side reactions,” he says. Another advantage of ICPL (compared with iTR AQ) is that the quantitation is done in the MS mode rather than in the MS/MS mode, significantly reducing analysis time as well as the amount of data acquired. Another relative newcomer is the ExacTag technology available through PerkinElmer. ExacTag also uses isobaric mass tags—similar to iTR AQ—but it labels proteins at either amines or thiols with a peptide rather than a small molecule; its reporter MS/MS ions fall between 343 and 475 Da. This method’s major advantage is that it offers the ability to process up to 10 samples simultaneously. When Perkin Elmer developed the ExacTag technology, “we were really interested in taking protein expression profiling to the pharmacologists,” says Banks. To generate kinetic plots or dose– response curves, these scientists need the higher multiplexing capability. “You can’t really generate an IC50 from two to four data points,” Banks explains. The final method for chemical tagging after cell lysis works slightly differently from the others. Rather than adding a chemical tag to the protein, 18O labeling incorporates its isotope directly into the backbone of the amino acid chain. The original concept was introduced to proteomics by Catherine Fenselau and colleagues at the University of Maryland and is quite simple: during trypsin cleavage, one of the samples is digested in 18O-labeled water while the other
is digested in “normal” 16O water (3). Trypsin incorporates the oxygen atoms into two positions in the terminal carboxy groups of the cleaved peptides. A kit for 18O labeling is currently available from SigmaAldrich. The method that the company recommends is very similar to the original concept, but its researchers found that digestion and labeling are best carried out in separate trypsincatalyzed steps. “You get much better results and much better efficiency,” explains Jennifer Williams of Sigma-Aldrich.
Chemical tagging during cell culture
SILAC, an extremely popular labeling method, steps back one level in the proteomics workflow and actually incorporates labeled amino acids during cell culture. “If people are growing cells, I would always suggest SILAC because you are growing the isotopes into the proteins,” says Asara. Therefore, “you always have 100% labeling efficiency,” he explains. In SILAC, originally introduced by Matthias Mann, now at the Max Planck Institute of Biochemistry (Germany), two cell-culture populations are grown under identical conditions, except that one is supplemented with one or more labeled amino acids and the other with the nonlabeled versions (4). After five or six doublings (for most cell lines), the natural metabolic chemistry of the cells has fully incorporated the amino acids into proteins. Kits for SILAC experiments are available from two sources. Invitrogen offers a variety of kits that are either general or targeted to specific subproteomes. “What we’ve been trying to do is create a flexible spectrum of reagents that allow different scientists to focus and target their experiments,” says Mike Kunis of Invitrogen. Each kit includes cell-culture media and 13C-labeled Lys (13C-, 15N-labeled Arg and 13C-, 15N-labeled Leu are also available optionally); the specialized kits also include lysis buffers and purification resins. Recently, Pierce Biotechnology (now a part of Thermo Fisher Scientific) also introduced SILAC kits that include media and 13C-labeled Lys. Pierce also sells 13C-, 15N-labeled Arg separately. For the do-it-yourselfers out there, labeled amino acids can also be obtained directly from Cambridge Isotope Laboratories without the added kit components. “The use of stableisotope-labeled amino acids in cell culture has become fairly routine; however, the use of stable isotopes in proteomic research will continue to expand,” says Tasha Agreste of Cambridge Isotope Laboratories. “We supply a large selection of amino acids and reagents that support the development and use of MS-based technologies in proteomics.” The company offers a complete listing of stable-isotope-labeled amino acids, including specifically labeled amino acids as well as (13C), (13C/15N), and (13C/15N/D) labels of Lys, Arg, Leu, Ser, Tyr, and Met.
Absolute quantification
Sometimes researchers need to know the exact concentration of a given protein, and the typical way to do this is through AQUA, a technique developed by Steve Gygi and colleagues S e p t e m b e r 1 , 2 0 0 7 / A n a ly t i c a l C h e m i s t r y
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at Harvard Medical School (5). AQUA Table 3. Selected absolute quantification reagents.1 is a variation on isotope dilution in Product Company Application notes which a known quantity of a labeled synProtein AQUA Sigma-Aldrich Delivered as a lyophilized power in 5 × 1 nmol aliquots; thetic peptide is spiked into the sample custom pep800-325-3010 >95% purity by HPLC; quantified by amino acid analysis and compared with the native version. tides www.sigmaaldrich.com In contrast to other methods, “AQUA Custom Heavy- Thermo Scientific Delivered in solution in 5 × 2 nmol aliquots; 97% purity; is a different technique in that it is not Peptide AQUA 866-984-3766 quantified by amino acid analysis a global labeling technique,” says Dale www.thermo.com Peluso of Sigma-Aldrich. “It’s more for Custom Heavy- Thermo Scientific Delivered as a lyophilized powder containing a minimum of validation, because you need to know Peptide Basic 866-984-3766 100 μg of peptide; 90% pure; meant for large-scale screenthe protein of interest that you are going www.thermo.com ing experiments; much cheaper than AQUA peptides after,” he adds. 1 Some companies may offer similar products not listed here. Contact the vendors for their full product lines. Because the protein of interest is different for each lab, AQUA peptides are only available through custom synthesis. Thermo Fisher to do a screening on a huge number of proteins to confirm Scientific offers two versions of their custom HeavyPeptides. data,” says Louette. The AQUA version is 97% pure, quantified by amino acid Sigma-Aldrich offers its own version of high-purity AQUA analysis, and supplied in solution. “We decided that the best peptides. Currently, the company only takes custom orders, way to deliver the product to maintain accuracy and precibut it hopes to develop a line of off-the-shelf products evension on the quantitation data was to deliver it in solution,” tually. “If someone discovers a novel biomarker and comes says Joel Louette of Thermo Fisher Scientific. The company up with a method to quantitate it, AQUA peptides for that also offers a line of peptides called Basic, which are only 90% particular biomarker are going to become very valuable,” says pure and are delivered as a lyophilized powder but are much Peluso. “It will probably become less and less custom as the cheaper than the AQUA line. “The Basic kit is really designed demand rises.”
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Researchers see some room for improvement on the currently available offerings. “The more the companies improve the isotope purity, the better the theoretical dynamic range is going to be,” says Andrews. “But to get the much higher purity reagents, the cost goes up exponentially, so I’m not sure that’s practical,” he concedes. Asara would like to see an improvement in protocol development to go along with these reagents. “You need to accommodate your whole customer base,” he says. For example, he thinks the companies should offer specific instructions for people working with each type of mass spectrometer as well as separate protocols for researchers analyzing each type of tissue matrix. Regardless, the quantitative MS proteomics toolbox is obviously overflowing, so much so that the field has already left 2DE in the dust. “When you look at how people do quantitation, it’s pretty much usually done through a mass spectrometer these days,” says Banks. Andrews agrees. “It’s an exciting time in proteomics because of all these new capabilities for MS quantification,” he says. “We’ve come a long way, and I expect to see further exciting developments in the next couple of years.” Jennifer Griffiths is an associate editor of Analytical Chemistry.
www.acdlabs.com/IntelliXtract/
References (1) (2) (3) (4) (5)
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Gygi, S. P.; et al. Nat. Biotech. 1999, 17, 994–999. Schmidt, A.; et al. Proteomics 2005, 5, 4–15. Yao, X.; et al. Anal. Chem. 2001, 73, 2836–2842. Ong, S. E.; et al. Mol. Cell Proteomics 2002, 1, 376–386. Stemmann, O.; et al. Cell 2001, 107, 715–726.
