Labeling Technologies for Quantitative Protein Expression Analysis

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1 Innovative Solutions for Quantitative Protein and Biomarker Research Labeling Technologies for Quantitative Protein Expression Analysis Understanding Disease States via Quantitative Protein Expression Analysis

2 New standards for quantitative protein expression analysis The key to understanding proteins and their behavior in biological systems lies in dramatically improving a broad and deeper analysis of the proteome. Researchers require new standards in protein expression analysis to identify and quantitate crucial proteins involved in disease and altered phenotypes, diagnostic biomarkers, and targets for therapeutic intervention. Applied Biosystems offers an integrated approach supplying everything you need for quantitative protein expression analysis (Figure 1) in one comprehensive platform of innovative products. Easily identify disease biomarkers Accurately identify drug targets Seamlessly resolve disease phenotypes PROTEIN EXPRESSION ANALYSIS WORKFLOW A. Cells B. Pathways C. proteins Diseased Normal Vimentin [Homo sapiens] beta-actin [Homo sapiens] aldolasea [Homo sapiens] 2-phosophopyruvategi/ gi/28336 gi/28614 hydratase alpha-enolase Vimentin [Homo sapiens] beta-actin [Homo sapiens] aldolasea [Homo sapiens] [Homo 2-phosophopyruvatehydratase alpha-enolase sapiens] gi/ gi/ gi/28336 gi/28614 Vimentin [Homo sapiens] beta-actin [Homo sapiens] aldolasea [Homo sapiens] [Homo 2-phosophopyruvate- sapiens] gi/ CV 9% gi/ CV 11% gi/28336 CV 10% gi/28614 CV 12% hydratase alpha-enolase [Homo sapiens] gi/ CV 9% CV 11% CV 10% CV 12% CV 9% CV 11% CV 10% CV 12% * * * * * * * * Glyceraldehyde-3-phospate peroxiredoxin 1 unnamed [Homo sapiens] unnamed [Homo sapiens] Dehydrogenase * [Homo sapiens] gi/ * gi/31170 * gi/28940 * [Homo Glyceraldehyde-3-phospate sapiens] gi/31645 peroxiredoxin 1 unnamed [Homo sapiens] unnamed [Homo sapiens] Dehydrogenase [Homo sapiens] gi/ gi/31170 gi/28940 [Homo Glyceraldehyde-3-phospate sapiens] gi/31645 peroxiredoxin unnamed [Homo sapiens] unnamed [Homo sapiens] CV 10% Dehydrogenase CV 16% [Homo sapiens] gi/ CV 14% gi/31170 CV 9% gi/28940 [Homo sapiens] gi/ N=5 2.5 CV 10% CV 16% CV 14% CV 9% 5.0 N=5 2.5 CV 10% CV 16% CV 14% CV 9% N=5 * * 2.5 * * * * * * Heat shock 70kDa protein 8 T-complex protein 1 unnamed [Homo sapiens] unnamed [Homo sapiens] [Homo sapiens] gi/ * [Homo sapiens] gi/31170 * gi/31092 * gi/24485 * Heat shock 70kDa protein 8 T-complex protein 1 unnamed [Homo sapiens] unnamed [Homo sapiens] [Homo sapiens] gi/ [Homo sapiens] gi/31170 gi/31092 gi/24485 Heat shock 70kDa protein 8 T-complex protein 1 unnamed [Homo sapiens] unnamed [Homo sapiens] CV 19% [Homo sapiens] gi/ CV 13% [Homo sapiens] gi/31170 CV 18% gi/31092 CV 11% gi/24485 CV 19% CV 13% CV 18% CV 11% CV 19% CV 13% CV 18% CV 11% * * * * * * * * * * * * Figure 1: Integrating proteomic workflows into quantitative analytical platforms. Identify differences in diseased versus normal cells (A) and their role in signal transduction pathways (B) by comparing the relative abundance of individual proteins (C).

3 Tools to Easily Perform Quantitative Protein Expression Analysis Quantitative protein expression analysis protocols rely on parallel analysis of samples to identify proteins that are differentially expressed. By applying simple protein fractionation protocols, you can seamlessly integrate robust quantitative analysis platforms into your workflows. Whether you choose the high fidelity of SILAC technology, or the chemical labeling flexibility of the Cleavable ICAT or itraq reagents, our quantitative protein expression tools enable you to analyze and identify protein expression changes from a variety of sample sources (Figure 2). Achieve high-fidelity protein labeling with SILAC Protein ID and Quantitation Kits Applied Biosystems combination of metabolic and chemical labeling technologies provides you with the flexibility to address the most difficult quantitation problems with confidence. Our integrated approach to quantitative protein expression analysis offers: Simple and easy-to-use workflows Confident and accurate identification and quantitation of protein expression differences in cell culture, tissue, or serum samples Broad protein and proteome coverage including post-translational modifications, membrane proteins, and biomarkers Identify and quantify membrane and low-abundance proteins with Cleavable ICAT reagents Analyze multiple proteins simultaneously using itraq reagents PROTEIN SOURCE CELL TISSUE SERUM EXPRESSION TOOL SILAC kits Cleavable ICAT reagents itraq reagents Figure 2: Perform quantitative protein expression analysis with SILAC, Cleavable ICAT, or itraq reagents Applied Biosystems offers quantitative protein expression tools that enable you to identify protein expression changes from a variety of sample sources to answer any biological question. See Table 1 to help you choose the quantitative protein expression analysis tool that is best for you.

4 Chemical labeling flexibility Analyze Multiple Samples Simultaneously Using itraq Reagents The ability to simultaneously analyze multiple samples readily enables important research studies such as those involving dosing, temporal investigations, pooling large sample sets for biomarker elucidation, and absolute quantitative determination of target proteins. itraq reagents are a multiplexed set of four or eight isobaric (same mass), amine-specific tags that yield labeled peptides, which are easily quantitated using mass spectrometry (MS). The simple peptide-based itraq workflow eliminates many of the restrictions and difficulties presented by other strategies, allowing protein coverage that includes post-translational modifications, affinity pull-downs, and membrane proteins. By combining itraq reagents and Applied Biosystems/MDS SCIEX instrumentation and application-specific software, such as ProteinPilot software, you ll achieve the meaningful, highly accurate results that you need to be confident in your quantitative protein expression analysis (Figure 3). Achieve identification and quantitation of multiple samples simultaneously Maximize protein/proteome coverage Increase statistical confidence and relevance quantitate up to eight samples at a time itraq TM Reagents Quantitation Figure 3: 4800 Plus MS/MS of *VAAHAVVAR peptide from Ovotransferrin showing simultaneous identification and relative quantitation of eight samples labeled with itraq reagents Identification R A V V A H A A V 70 % Intensity y1 40 b2 b3 b4 b9 b y5 b5 b7 b8 b1 y2 y3 y4 y6 y8 y7 10 y9 y Mass (m/z)

5 Improve Quantitative Protein Expression Using Cleavable ICAT Reagents and Mass Spectrometry Cleavable ICAT reagents are cysteine-specific, affinity-enabled mass tags that provide a powerful mass spectrometry based method for separating and quantitatively analyzing protein expression differences from complex samples. Cleavable ICAT reagents allow you to readily identify and quantify important classes of proteins, such as membrane and low-abundance proteins, that are often difficult to analyze by traditional methods. Cleavable ICAT reagents and workflows are easily integrated with Applied Biosystems/MDS SCIEX MS instrumentation and application-specific software for rapid unattended quantitation and identification of biologically significant proteins (Figure 4). Achieve cysteine-specific protein labeling with precise MS quantitation Identify membrane and low-abundance proteins Integrate workflows into analytical platforms and software IL-4 treated cell lysate 400 µg total protein Reduce with TCEP Label with H-ICAT reagent Combine samples SDS-PAGE In-gel tryptic digestion of excised gel bands Digested peptide extraction Avidin affinity/tfa cleavage LC/MS/MS for protein ID and quantitation Control cell lysate (no treatment) 400 µg total protein Reduce with TCEP Label with L-ICAT reagent A MW (kda) Lanes Protein bands In-gel tryptic digest B 60S ribosomal protein exhibits 50% downregulation upon IL-4 treatment of cell lysate Untreated IL-4 treated cell lysate C Report generated with protein identification and quantitation Mark 12 Unstained Protein Standard 2. ACHN, before labeling with ICAT 3. ACHN, after labeling with ICAT Figure 4: Quantitative protein expression comparison of two cell states using Cleavable ICAT reagents and MS analysis Cytokine regulation of protein expression in IL-4 treated and control ACHN cells is evaluated via Cleavable ICAT reagents and a 1D-PAGE approach. In this method, proteins are released by cell lysis, and each sample is labeled with either light or heavy ICAT reagents. Once labeled, samples are combined and fractionated on a 1D SDS-PAGE system. A single band corresponding to the target MW range of kda is excised (A). Subsequent in-gel trypsin digestion, extraction, and affinity isolation of the ICAT labeled peptides are performed prior to MS and MS/MS analysis to quantitate and identify proteins regulated by IL-4 treatment (spectrum in (B) and report in (C).

6 High fidelity labeling Explore Cellular Phenotypes Using SILAC Kits and Mass Spectrometry SILAC protein labeling kits allow you to study perturbations in pathways that regulate the expression of crucial proteins and identify those that lead to altered phenotypes or deleterious effects. With SILAC technology,1 you ll combine current cell culture workflows with traditional fractionation protocols, such as chromatography or gel separation techniques, to achieve detailed quantitative analysis of cellular function and dysfunction mechanisms along with their possible protein origins. Integrating the SILAC labeling strategy with the Applied Biosystems/MDS SCIEX family of MALDI TOF/TOF Analyzers results in an even more empowering dimension for proteomic and cellular dysfunction analysis via MS (Figure 5). Now you can easily explore cellular phenotypes by performing detailed quantitative analysis of post-translational modifications, low-abundance proteins, phosphoproteins, membrane proteins, gene knockdown experiments, and much more. Easily study cellular pathways that regulate protein expression Reliably analyze post-translational modifications and low-abundance proteins Seamlessly integrate cell culture workflows into rigorous analytical platforms SILAC WORKFLOW SILAC Protein ID and Quantitative Kits STEP 1 STEP 2 Grow duplicate cultures in SILAC media with isotopically distinct amino acids (AA). If needed, apply stimulus (e.g., RNAi) to culture grown in SILAC media. Harvest and mix cells. SILAC heavy AA SILAC light AA STEP 3 STEP 4 STEP 5 e cells using SILAC lysis buffers, run lysate on NuPAGE 1D gel, excise gel band, and trypsinize. Submit samples to MS operator for data analysis. Obtain list of proteins identified with relative abundances. Figure 5: SILAC Protein ID and Quantitation Kits provide reagents and protocols for metabolic protein labeling and analysis with mass spectrometry. SILAC reagent workflows begin by labeling parallel cell cultures with either a light or heavy amino acid for six doublings (Step 1) to ensure near-100% metabolic incorporation. After labeling, one cell population may be challenged experimentally. Cells are then harvested and mixed 1:1 (Step 2), followed by lysis and fractionation of proteins by SDS-PAGE (Step 3). Gel bands are submitted for MS analysis (Step 4). Finally, easy-to-interpret results from Applied Biosystems/ MDS SCIEX MALDI TOF/TOF Analyzers list identified proteins complete with quantitative information (Step 5).

7 Time-saving protein expression analysis technologies save research dollars Many methods for analyzing proteins and proteomes require tedious and time-consuming evaluation of multiple tools and technologies, learning new workflows, and optimizing experimental conditions to meet your proteomic research goals. Applied Biosystems offers innovative quantitative protein expression analysis tools and technologies that save you time and money and simplify your proteomic research efforts. You ll spend less time optimizing and more time obtaining definitive and accurate results. In addition, you ll reduce your research costs by using fractionation techniques that are already available in your lab or core facility. Begin your protein expression analysis experiments today. Use Figure 6 to choose the technology that is best suited for your research goals. Eliminate time spent learning new workflows Decrease time required to optimize experimental conditions Reduce costs by using fractionation techniques available in your lab or core facility TABLE 1: Choose the protein expression analysis technology that best suits your needs Research objective SILAC ICAT itraq reagents reagents reagents Analyze proteins from tissues or serum 3 3 Analyze up to eight samples simultaneously 3 Analyze post-translational modifications 3 3 Perform sample fractionation by SDS-PAGE 3 3 Perform affinity enrichment of labeled peptides 3 Cell culture Tissue/serum Metabolic labeling Chemical labeling SILAC reagents Compare 2-8 samples Compare 2 samples itraq reagents ICAT reagents

8 From the leaders in quantitative proteomics Applied Biosystems is the leading provider of innovative solutions for quantitative proteomics. Our labeling chemistries are a perfect complement to the industryleading Applied Biosystems/MDS SCIEX mass spectrometry and LC platforms. Our total system solutions are optimized for your workflows to provide highest-confidence results for protein expression and protein biomarker discovery experiments. 4800Plus MALDI TOF/TOF Analyzer BIOiTRAQ II QS LC/MS/MS System MIDAS TRAQ LC/MS/MS System Reference 1. Ong, S. et al. (2002) Mol. Cell Proteomics 1: 376 For Research Use Only. Not for use in diagnostic procedures. Applied Biosystems, (AB) Design, itraq, BIOiTRAQ,and Applera are registered trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. MALDI TOF/TOF and MIDAS are trademarks of Applied Biosystems/MDS SCIEX, a joint venture between Applera Corporation and MDS Inc. SILAC is a trademark of Invitrogen Corporation. ICAT is a registered trademark of the University of Washington and is exclusively licensed to the Applied Biosystems Group of Applera Corporation. All other trademarks are the sole property of their respective owners Applera Corporation and MDS Inc. All Rights Reserved. Information subject to change without notice. Printed in the USA, 01/2007 Publication 114BR24-01 Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone Toll Free International Sales For our office locations please call the division headquarters or refer to our Web site at

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