EXPRESSION MORE PEPTIDES. MORE PROTEINS. MORE REPRODUCIBILITY. MORE QUANTIFICATION.

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1 A New Era in Protein Quantification [ systems BIOLOGY Solutions ]

2 [ systems BIOLOGY Solutions ] EXPRESSION MORE PEPTIDES. MORE PROTEINS. MORE REPRODUCIBILITY. MORE QUANTIFICATION. The Waters Expression E High Definition Proteomics System defines the new standard for rigorous protein quantification in bottom-up analysis. Designed for comprehensive quantitative and qualitative protein profiling, this total system solution combines high bandwidth UPLC/MS E data acquisition and Waters stringent protein ID technology Identity E with multi-peptide, label-free, protein quantification. Now life scientists can reproducibly report the maximum number of secure protein identifications together with stringent differential quantification and absolute protein concentration. The Expression E System comprehensively maps complex biological systems with greater statistical rigor than established MS-based strategies enabling a new era in biomedical research.

3 The Waters Expression E High Definition Proteomics System integrates Waters nanoacquity UPLC System with a Waters Q-Tof Premier or Waters SYNAPT High Definition Mass Spectrometry System with optimized sample preparation chemistries, statistically rigorous bioinformatics, optimized analytical methods, and comprehensive system training.

4 [ systems BIOLOGY Solutions ] THE WATERS EXPRESSION E HIGH DEFINITION PROTEOMICS SYSTEM The Expression E System has been specifically developed to address the analytical challenges inherent in contemporary proteomics including: n Biomarker discovery n Molecular diagnostics R&D n Identifying the component proteins in pull-down experiments n Protein flux analysis n Global protein profiling The Waters Expression E High Definition Proteomics System integrates a Waters nanoacquity UPLC System with a Waters Q-Tof Premier or Waters SYNAPT High Definition Mass Spectrometry (HDMS ) System with optimized sample preparation chemistries, statistically rigorous bioinformatics, optimized analytical methods, and comprehensive system training. The result is a sophisticated system solution that facilitates stringent proteomics discovery research with defined performance. MEETING THE PROTEOMICS CHALLENGE Proteomics is increasingly focused on biomarker discovery and understanding disease mechanisms at the molecular level to develop molecular diagnostics, targeted therapies, and personalized medicine. This necessitates identifying the components of complex mixtures that contain thousands of proteins, and quantitatively comparing them over a wide dynamic range. I told my students, You don t know how bad ion trap (proteomics) data can be until you have seen how good Expression E data is! A leading US academic and owner of four ion traps Traditional bottom-up proteomics analysis is frustrated by analytical incompleteness or under-sampling. This phenomenon is a systemic weakness of all LC/MS/MS systems operated in the serial, data directed analysis (DDA) mode. In contrast, the Expression E System employs a unique high bandwidth UPLC/MS E data acquisition strategy to consistently over sample complex peptide digests to deliver global Expression E datasets containing evidence for all peptides above the limit of detection. In differential proteomics (relative quantification), most contemporary approaches necessitate isotopic/isobaric labeling and analysis of a minority of (affinity selected) peptides associated with each protein contained within a sample. Conversely, the Expression E System does not require peptide derivatization to quantify the majority of tryptic peptides within a complex digest.

5 [ EXPRESSION E system ] GO BEYOND DATA-DIRECTED ANALYSIS As with any analytical technique, the validity of results are constrained by the quality of raw data. The Waters Expression E system combines UltraPerformance LC with MS E detection and industryleading dynamic mass resolution. Waters UPLC/MS E technology consistently over-samples complex digests for comprehensive peptide detection. This analytically-complete approach delivers protein identification with unmatched sequence coverage. Waters chromatographic advancements in UPLC, with rugged sub-2 µm Bridged Ethyl Hybrid (BEH) particle columns, represent the most technologically advanced solution for nanoscale separations of complex tryptic digests. UPLC enables separations with maximum resolution, speed, and sensitivity. The peak capacity and robustness of UPLC accentuates the specificity of the Expression E system s retention time prediction model, allowing the amino acid composition of peptides, identified from matching MS E spectra, to be corroborated. Likewise, Waters innovations in data-independent MS E have produced a revolutionary data acquisition technique. Waters premier orthogonal time-of-flight (TOF) mass spectrometers provide the highest dynamic mass resolution for LC/MS applications with 17,500 FWHM at 10 spectra/ sec facilitating outstanding peak definition and accurate mass measurement. UPLC/MS E acquires data with two rapidly alternating MS functions; the first contains exclusively low-energy mass spectra, the second is composed of mass spectra acquired at elevated collision energy (designated as MS E ). The resulting data set contains a comprehensive time-resolved record of all detectible precursor and product ions. Precursor and product ions are associated by both retention time alignment and peak shape, enabling even chimeric peptides and overlapping ion clusters to be successfully handled. The resulting time-aligned precursor and product ion mass lists are used for peptide/protein identification with Waters Identity E bioinformatics. The intensity of each peptide s (multiply charged) molecular ions are used for both relative and absolute quantification. Data stringency In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exiting new technologies has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality. M.R. Wilkins et al. Guidelines for the next 10 years of Proteomics. Proteomics Jan; 6 (1): 4-8. Data acquisition using UPLC/MS E.

6 [ systems BIOLOGY Solutions ] A NEED FOR CHANGE Too many papers that I receive and/or read in scientific journals have no evidence of any repeatability, much less reproducibility Thus, I suspect that > 50% of papers now being published in the proteomics field have data that was performed but once (n = 1), which makes the entire work unacceptable for publication. W.S. Hancock (Editor-In-Chief). An Analytical Chemist s Perspective. Journal of Proteome Research Vol. 6 (5), GLOBAL EXPRESSION E DATA SETS Global Expression E data sets provide a comprehensive record of all peptides detectable by ESI LC/MS within a sample and can be mined exhaustively for both qualitative and quantitative information, such as up- and down-regulated proteins, biomarkers, and the abundance of targeted proteins or peptides. Exact mass retention time (EMRT) signature files can be exported to public domain data repositories and third-party data analysis packages such as Rosetta Elucidator, Umetrics Simca-P or Spotfire, for orthogonal data correlation. STRINGENT DIFFERENTIAL QUANTIFICATION Multiple normalized global Expression E data sets are compared and contrasted in relative protein expression analyses. In all cases a minimum of three technical replicates are analyzed to facilitate statistically rigorous reporting of quantitative and qualitative results. EMRT signatures enable all detected peptides to be uniquely identified and enable any two global Expression E data sets to be differentially compared. The normalized intensity of each EMRT signature is indicative of the abundance of a specific, tryptic peptide. Each protein in a global expression data set is represented by multiple peptides. The relative abundance of any protein is calculated by comparing multiply-matched EMRT signatures (n 3), allowing limits of confidence to be determined. Hi3 ABSOLUTE QUANTIFICATION Absolute protein quantification conventionally requires co-determination of a unique peptide for each targeted protein, with its corresponding stable isotope labelled internal standard. Such isotope dilution methods remain the gold standard for absolute protein quantification and are essential in clinical diagnostic procedures. However, Waters high-three (Hi3) absolute quantification provides a rapid and cost effective intermediate strategy that enables pre-clinical trial validation of putative biomarkers. log10 (Average pg/ml) Hi3 Label-Free Absolute Quantification of Human Serum Proteins _Albumin 04_Transferrin 06_Alpha-2-macroglobulin 07_Alpha-1-antitrypsin 08_C3 Complement 09_Apolipoprotein A-1 Human Serum Proteins Human Serum Proteins 11_Alph-1-acid glycoprotein 12_IgM Total 13_Haptoglobin 14_IgG Total 16_Ceruloplasmin Hi3 methodology references the combined intensity of the multiply charged molecular ions for the three most abundant tryptic peptides of a quantitatively added Expression E protein standard with the observed Hi3 response for any identified protein in a complex digest mixture. The Hi3 peptide response for any protein is similar; therefore, the summed intensity of the Hi3 Expression E reference peptides may be used to estimate the molar amount of any protein present in a complex mixture. As a result, the Hi3 methodology is applicable to estimate the absolute concentration of any protein identified within an Expression E data set. Average Hi3 determined concentration (blue circles) and %RSD (blue whiskers) for 11 human serum proteins from 10 volunteers. Average expected concentrations (red circles) with minimum/maximum values (red whiskers), calculated using both ELISA and enzymatic assay data (Specialty Laboratories). The experimental Hi3 data show a high degree of similarity.

7 [ EXPRESSION E system ] MRM METHODS MADE EASY Waters Global Expression E data sets contain a comprehensive record of masses, charge states, ion intensities, and retention times of all precursor and fragment ions above the limit of detection. They contain all the necessary data to construct Multiple Reaction Monitoring (MRM) methods for high throughput target peptide analyses on triple quadrupole instruments. Optimum MRM transitions for unique peptides can be rapidly identified and incorporated into LC/MS/MS methods. From here, they are ported directly to one of Waters high performance tandem quadrupole MS systems, which share the same MassLynx Software, Z-Spray ion source, and UPLC technologies as the Waters Expression E High Definition Proteomics System. THE ULTIMATE PROTEOMICS PLATFORM The Expression E High Definition Proteomics System quantitatively and qualitatively maps complex biological digests with greater statistical rigor than established MS-based strategies, providing a secure foundation for biological discovery and medical research. SYSTEM COMPONENTS AND OPTIMIZED PERFORMANCE KIT The Waters Expression E System consists of: n Waters nanoacquity UPLC System n Waters SYNAPT HDMS System n Waters Q-Tof Premier Mass Spectrometer n Waters Expression E System Informatics n Waters Expression E Applications Kit n Waters Expression E System Manual and Documentation n Waters Expression E System Training Course The Expression E High Definition Proteomics System Applications Kit provides all of the materials required to easily perform the system performance verification and acceptance test. The kit includes Expression E System Standards, RapiGest SF Digest Reagent, nanoacquity UPLC BEH 1.7 µm columns, standard operating procedures, method templates, and reference data. PROTEIN Expression E : Label-Free Relative Quantification T rue Fold Change EXPRESSION E FOLD CHANGE EXPRESSION E FOLD CHANGE % ERROR Bovine Serum Albumin % Lactoperoxidase % Ribonuclease % Peroxidase C1A % Casein % Catalase % Carbonic Anhydrase % Glycogen Phosphorylase % Average = 4.6 % The fold change differences across two samples containing eight well characterized proteins in known amounts was determined using Waters Expression E Label-Free relative quantification protocol with an average error of 4.6%. These data compare very favorably with a recent 20-laboratory study of identical samples, employing all of the common commercial isotopic/isobaric labelling techniques, which exhibited an average error of 46%. Completeness Analytical incompleteness refers to a phenomenon where a technique used for the analysis of complex mixtures of peptides may only yield information for a fraction of relevant peptides in an single analytical run. M.R. Wilkins, et al. Guidelines for the Next 10 Years of Proteomics. Proteomics Jan; 6 (1): 4-8.

8 Sales Offices: Austria Australia Belgium and Luxembourg Brazil Canada China Czech Republic Denmark Finland France Germany Hong Kong Hungary India Ireland Italy Japan Korea Mexico The Netherlands Norway Poland Puerto Rico Russia/CIS / Singapore Spain Sweden Switzerland Taiwan UK US Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: For more information on the Waters Identity E approach, please refer to the Identity E High Definition Proteomics System brochure, available on (Literature Code EN). Waters, nanoacquity UPLC, UltraPerformance LC, and UPLC are registered trademarks of Waters Corporation. The Science of What s Possible, Expression E High Definition Proteomics, Q-Tof Premier, High Definition Mass Spectrometry, HDMS, MassLynx, Z-Spray, and RapiGest are trademarks of Waters Corporation. All other trademarks are property of their respective owners Waters Corporation. Printed in the U.S.A. January EN TL-AC