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Reprint from D a i l y B i o t e c h U p d a t e s . . . w w w. g e n e n g n e w s . c o m Volume 24, Number 8 April 15, 2004 DRUG DISCOVERY Mass-Defect Tagging for Proteomic Analysis Tutorial: Broadened Applicability of Differential Display Mass Spectrometry Luke V. Schneider, Ph.D., Michael P. Hall, Ph.D., and Michael M. Brasseur ass spectrometry has the ability to analyze DNA, peptides, proteins, and small molecule drugs in automated, high-throughput assays, enabling and/or improving such applications as protein target identification, validation, ADMET assays, and others. The unique advantage of MSbased assays is the ability to gain more molecular-level information about an analyte, such as posttranslational modifications, and thus add more clinical relevance to the assay. Protein differential-display analysis has proved useful for the determination of drug mechanisms and for assessing toxicological potential during preclinical M Figure 1. Mass-defect periodic table screening studies. Protein microarray analysis has been used to find protein biomarkers of ovarian cancer in plasma. With the average mammalian cell expressing anywhere from 10,000 to 40,000 proteins, however, many are only subtly different from each other. Good differentiation techniques are thus essential to the generation of high-quality data. Like many measurements conducted in biology, MS measurements are often ratiometric rather than absolute. In MS, quantitative comparisons between samples, or even between spectra of the same sample, are not reliable without the use of stable isotope techniques. Several proteomic techniques, such as [16/18O]water hydrolysis of proteins, global internal standard technology, and isotopecoded affinity tags, have been described in the literature. These differential display techniques allow direct quantitative comparison of relative protein expression between two or more tagged samples, by comparing the relative abundance of stable isotope versions of the same tag. The advantage of these differential display methods is that they are relatively inexpensive, quick, and accurate (<10% standard deviation). The problem with these techniques is that they require separation of tagged species from untagged species prior to MS detection, to avoid false positive and negative results. For this reason, differential display mass spectrometric methods have not been applicable to chip-based methods (e.g., affinity protein or DNA microarrays). By digesting the proteins bound to an antibody on a protein chip, the antibody is also digested, producing a confusing array of background peptides in the mass spectrum. Similarly, DNA oligomers of different lengths due to incomplete PCR amplification reactions and/or nonstringent binding conditions in DNA microarrays, can yield spurious signals in the mass spectrometer that may confound the identification and quantification of the peaks of interest. By adding a mass-defect element to a pair of stable-isotope tags, Target Discovery (Palo Alto, CA) aims to address this limitation. These isotope-differentiated binding-energy–shift tags (IDBEST™) shift the peaks of all the tagged species by about 0.1 Da, allowing software to discriminate tagged from untagged species directly in the mass spectrum and thus eliminating the need for affinity cleanup of the tagged samples. The mass defect is related to the nuclear binding energy released upon formation and stabilization of the nucleus of a given element. Chemical-noise peaks in mass spectra predominantly arise from unlabeled peptide fragments. These fragments are composed of combinations of C, H, N, and O atoms, which exhibit minimal mass defects (Figure 1), therefore, peaks resulting from biomolecules have an intrinsic regular spacing in the mass spectrum. Utilizing a tag with an element containing a significant mass defect (e.g., between Br and Eu in the periodic table) shifts the mass of the tagged biomolecules off the intrinsic spacing of the untagged biomolecules (Figure 1). Bromine and iodine are good mass-defect elements in that they are easily incorporated into organic tags. Bromine is particularly advantageous since it has a lower mass than iodine and has a nearly equivalent natural abundance of its two stable isotopes (79Br and 81Br.). It is noteworthy that, in high-resolution mass spectrometers, the space between the single- and higher-charge states of biomolecules (i.e., negligible–mass-defect species) leave a window of nearly 0.4 amu unoccupied in nearly every amu of the mass spectrum. Therefore, it is posFigure 2. Mass spectrum and deconvolved spectrum showing sible to incorporate up to four mass-defect peaks in an IDBEST™ differential display applicamass-defect atoms into an tion IDBEST reagent before substantial overlaps with other biomole- tides are shifted from the unlabeled pepcules are encountered. Since each mass- tides, the relative ratios of light- and heavydefect element shifts the peak of the chain IDBEST peptides can be determined, tagged species by an additional –0.1 and, thus, differential expressional differamu, a 1–mass-defect IDBEST tag can be ences can be precisely determined. Figure 2 shows an example in which resolved from a 2–mass-defect IDBEST BSA samples were labeled separately with tag, and so on. light and heavy versions of an IDBEST Protein Differential Display tag and mixed in equal portions in a pool The mass defect in combination with of unlabeled E. Coli cytosolic proteins. isotope-paired reagents can be used to The entire mixture was trypsinized and determine expressional differences between analyzed on an Applied Biosystems 4700 control and perturbed samples (e.g., healthy MALDI-TOF MS Proteomics Analyzer. versus diseased). In this case, the complex Panel A shows the raw mass spectrum protein mixtures are labeled separately in the region surrounding the [286-97] with light or heavy chains of IDBEST. The tryptic fragment (labeled monoisotopic samples are mixed, and the target pro- mass of 1,682.64 Da). The peaks corretein(s) of interest enriched by affinity sponding to the IDBEST peptide are seen purification, such as on a microarray. as shoulders shifted to the left of the abunThe resulting spot is digested to peptides dant chemical noise. and directly analyzed by MALDI mass specPanel B shows the mass-defect spectrometry. Since the IDBEST-labeled pep- trum after deconvolution by the Target sequencing by a method called inverted mass ladder sequencing (IMLS™). IMLS is a variant of top-down sequencing, in which an N- or C-terminal PST is generated from the intact protein by fragmentation in-source in an inexpensive MS. IMLS involves labeling the terminus of a protein with a mass-defect tag that allows assembly of a PST by mass addition of fragment ions starting with the unique mass of the chemical tag. As described above, the fragmentation of whole proteins generates a multitude of fragFigure 3. N-Terminal top-down protein-sequencing application ment ions (“chemical noise”), of mass-defect tags showing the raw protein-fragmentation giving rise to peaks at nearly spectrum, the deconvolved mass-defect spectrum showing the Br-doublets, and the final peak-paired sequencing spectrum every mass position in the spectrum with an average Discovery software. The chemical noise sequence-dependent peak spacing of has been effectively eliminated and the 1.000464 amu. For example, myoglobin isotope series of IDBEST-labeled tryptic was labeled with a bromine-containing peptide are evident. Peak 1 corresponds to IDBEST tag, and the published sequence the monoisotopic (12C/79Br) peptide. of myoglobin (GLSDGE) through six Peak 2 corresponds to an overlap of both residues was recovered from the IMLS the second 13C isotope of the 79Br light spectrum generated in an Applied BiosysIDBEST tag, the 12C isotope of the 81Br tems Mariner ESI-TOF MS (Figure 3). light IDBEST tag, and the 79Br version of The IMLS software both deconvolves the the heavy IDBEST tag. Peak 3 corresponds mass-defect spectrum and automatically predominantly to the 81Br heavy IDBEST- determines the protein sequence by mass tagged peptide. addition to the known mass of the tag. While visually confusing, these isotopic Conclusion distributions are easily resolved by the software into simple light and heavy Mass-defect tags provide a powerful IDBEST-tagged contributions. The ratio method for enhancing the utility of MS in of light to heavy chain in this example is proteomic analysis, both for protein differ1.04, which is very close to the expected ential display in affinity microarrays and value of 1 of the known 50:50 mixture. protein sequencing by IMLS. This approach may also have utility in many Mass-Defect Tags in Protein other biomolecular mass spectrometric Sequencing Target Discovery has also successfully applications, such as identification of utilized mass-defect tags for protein splice variants from DNA microarrays and the sequencing of oligosaccharides. The key advantage of a differential display strategy based on the mass defect is preservation of the relative abundance of each isotope peak, because these are shifted away from any chemical noise. In addition, this shift potentially eliminates the need for prior separation (e.g., affinity purification of the tagged species). Incorporating a nonextendable base containing one or more mass-defect elements into DNA sequencing methodologies should allow discrimination of the resulting mass spectral sequence ladders from exogenous DNA in the sample. Furthermore, it should be possible to analyze the sequencing ladders for all four bases simultaneously if a different number of mass-defect elements are incorporated into each terminal base. Another advantage of mass-defect tagging is that it potentially allows for the simultaneous discrimination of more than two molecules. Just as a single massdefect element tag can be discriminated from unlabeled chemical noise, tags containing different numbers of mass-defect elements can be discriminated from one another within an amu. Therefore, mass-defect tags may allow up to five different species with the same nominal mass to be discriminated in the mass spectrometer before hitting the double– charge-state limit. This suggests that mass-defect tags may also extend the number of possible tags that can be discriminated simultaneously in combinatorial chemistry and high-throughput screening applications where mass tags are used. GEN Luke V. Schneider, Ph.D., is CSO, Michael P. Hall, Ph.D., is senior scientist, and Michael M. Brasseur is director of business development, at Target Discovery (Palo Alto). For more information, contact Michael Brasseur. Phone: (650) 812-8132. E-mail: [email protected]. Website: www.targetdiscovery.com.