SYSTEM SOLUTIONS FOR PROTEOMICS

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1 SYSTEM SOLUTIONS FOR PROTEOMICS Is your proteomics solution capable of: Fast, easy, and confident identification of potential protein drug targets or biomarkers? Comprehensive characterization of proteins and post-translational modifications? A simple route to the highest quality MALDI and electrospray data? Impressive search specificity and speed? Systematic extraction of more qualitative and quantitative information from complex digests? Complete system solutions from chemistry and UPLC to mass spectrometry and bioinformatics? Complete system solutions for protein discovery Proteomics research is increasingly focused on elucidating disease mechanisms and identifying protein biomarkers for diagnostics and therapeutic monitoring. Whatever your research objectives, Waters can provide you with unique high performance research tools that allow you to fully investigate proteomics samples. Our discovery systems for proteomics feature state-of-the-art technology from efficient sample preparation, HPLC and Ultra Performance LC separations to high performance research grade MALDI and electrospray mass spectrometers all fully integrated with bio-informatics. Combined with Waters worldwide services and support, you re assured of complete confidence in your systems and your results. Advance your proteomics capabilities with: Protein separations and pre-fractionation Rapid, confident protein identification High performance protein characterization Sensitive, specific protein modification analysis Label-free protein quantitation and biomarker discovery Protein interaction analysis

2 Your partner in advancing life science research Waters is dedicated to providing solutions for life science research. Our integrated discovery systems are the product of more than 30 years of commitment to excellence in LC, mass spectrometry, separations science, chemistries and informatics. We are committed to developing the tools you need for today s demanding post-genomic discovery science. Protein Separations Alliance Bioseparations Systems BioSuite Columns Protein Interactions LCT Premier Mass Spectrometer Q-Tof Premier Mass Spectrometer A complete proteomics solution Waters pioneered the routine characterization of proteins by exact mass measurement with our award-winning Micromass Q-Tof instruments. It is proven that exact mass measurement is a key metric in proteomics to improve the reliability of databank search results. When this metric is applied to peptide de novo sequencing it can also greatly enhance the confidence in individual amino acid assignments. As proteomics data sets increase in complexity so does the requirement for a powerful additional filter, such as exact mass measurement, in providing confident, validated results. IBM, our strategic life science partner, provides the most powerful bio-computing platforms available for quickly distilling large datasets. Today our acclaimed separations science portfolio, innovative mass spectrometers and best in class bio-informatics deliver Critical Mass in Proteomics for today s investigators. Waters analytical systems now allow proteins of biological significance to be isolated, identified and quantified with greater sensitivity, selectivity and speed than ever before. Sample Preparation MassPREP Station MassPREP PROtarget Plates MassPREP Glycoanalysis Kit MassPREP Matrices RapiGest SF Reagent Peptide Separations nanoacquity UPLC System nanoacquity UPLC Columns NanoEase Columns BioSuite Peptide Analysis Columns MALDI/MS and PSD MALDI micro MX Mass Spectrometer PSD MX Technology MALDI and ESI MS/MS Q-Tof Premier Mass Spectrometer Q-Tof micro Mass Spectrometer Mass spectrometry has been part of the chemist s toolkit for identifying small molecules since the beginning of the 20th century. But for many years, being able to make accurate measurements of the molecular masses of large proteins was a dream for chemists. Mass-Informatics and Bio-Informatics MassLynx Software ProteinLynx Global SERVER Protein Expression Informatics Professor Astrid Gräslund, 2002 Nobel Prize in Chemistry Citation

3 Integrated proteomics Waters ProteinLynx Global SERVER is a completely integrated, scalable bio-informatics platform for MS-based protein analysis. Its unique architecture provides an environment for protein identification and characterization in MS-based studies through an intuitive, integrated browser. Samples and mass spectral data are systematically catalogued and managed through each data acquisition stage followed by automated processing, interpretation and reporting with the ground-breaking MS-WorkFlow Manager. Key features of ProteinLynx Global SERVER include: Comprehensive toolset For protein identification, characterization and relative quantitation. Integrated project and sample management for MALDI-TOF-MS, PSD MX and MALDI/ESI MS/MS analyses. Support for 2D gel and 2D LC workflows. MS-WorkFlow Unique capability enables multiple algorithms (e.g. MaxEnt 3 processing, databank searching, AutoMod, MassSeq and BLAST ) to be chained for automation Combined power Fully integrated searching/reporting with ProteinLynx and MASCOT (Matrix Science) Maximum confidence ProteinLynx Global SERVER exploits the specificity of exact mass data to maximize confidence in results from databank searching and de novo sequencing Throughput Tailored IBM e-server solutions to meet your demands Integrated discovery XML file format and LIMS integration further expands your analytical potential ProteinLynx Global SERVER rapidly interprets high-resolution MS and MS/MS data and reports statistical results in a convenient browser for easy review. This display shows results of a combined ProteinLynx Global SERVER 2.2 and MASCOT databank search, where the identified protein is annotated on an imported 2D gel image.

4 Protein identification 2D gel electrophoresis is a traditional strategy for the separation and relative quantitation of proteins from very complex mixtures as it allows up-regulated or down-regulated proteins to be detected. However, the process is labor-intensive, because once protein spots are excised, they require digestion and preparation for MS analysis. Waters Protein Identification System eliminates these timeconsuming steps by automating sample digestion/preparation and MALDI-TOF-MS and PSD analysis. Streamlined, high throughput protein identification Automated, easy-to-use and fast, the Waters Protein Identification System is the ideal solution for rapid, confident protein identification from large sample sets because it provides: Automated sample digestion and preparation The MassPREP Station automatically and effortlessly processes PAGE and LC-separated samples for MALDI and ESI analysis. Significantly increased detection limits MassPREP PROtarget plates increase detection limits up to 10 times and enable analysis of previously unidentifiable samples. Automated sequence analysis Revolutionary PSD MX technology provides simultaneous sequence analysis of peptides in a mixture for faster experiments and more protein identifications. Waters Protein Identification System incorporates the MassPREP Station, MassPREP target plate technologies and the Micromass MALDI micro MX mass spectrometer with MassLynx and ProteinLynx Global SERVER software. Integrated discovery Identification and reporting of proteins from ProteinLynx and MASCOT databank searches using MS (PMF) and PSD MX data. Higher performance The high resolution MALDI micro MX MALDI-TOF Mass Spectrometer is engineered for exceptional performance, reliability and ease-of-use. Effortless operation Automated data acquisition with a simple wizard-driven setup makes MALDI more accessible to users with limited MS experience. Knowledge sharing Gel images and spot lists imported into the ProteinLynx Browser are automatically annotated with database search results and seamlessly exported via XML to software packages for subsequent evaluation. PSD MX analysis provides the additional sequence specificity required for unambiguous protein identification, when peptide mass fingerprinting (PMF) leads to inconclusive assignment of the parent protein. The PMF and PSD MX data shown here is from analysis of Hemoglobin A alpha I chain.

5 Protein identification and protein characterization by MS/MS Protein identification by PMF can be hindered if the number of peptides present from a single protein is low, if protein modifications are prevalent or the proteins and species of interest are not well represented in databases. In such cases, electrospray (ESI) or MALDI MS/MS analyses with Waters Micromass Q-Tof mass spectrometers produce the highest quality sequence data for more successful protein identification and characterization. Sensitive benchtop protein characterization Perform protein identification with the added specificity afforded by exact mass MS/MS in a highly sensitive LC/MS/MS configuration with the benchtop Waters Protein Characterization System. Featuring the Q-Tof micro mass spectrometer, this system provides unique capabilities including: Ultra Performance Peptide Separations Waters nanoacquity UPLC System and bridged ethylsiloxane/silica hybrid (BEH) 1.7 μm particles provide unmatched resolution and peak capacity for peptide separations. High performance sequence analysis Q-Tof technology with the NanoLockSpray source enables exact mass MS and MS/MS data to be obtained for maximum confidence in results. Automated analysis Wizard-driven, intelligent Data Directed Analysis (DDA ) and patented Precursor Ion Discovery (PID) analysis offer automated sequence and modification analysis of peptides. Integrated discovery Achieve integrated system control with the market-leading MassLynx 4.0 software platform and ProteinLynx Global SERVER bioinformatics. Protein sequence characterization Enhance protein sequence coverage with Q-Tof technology and ProteinLynx Global SERVER bioinformatics. This powerful duo lets you confidently identify: Databank proteins with maximum confidence using exact mass MS/MS data Missed and non-specific cleavages, multiple amino acid substitutions and multiple peptide PTMs with AutoMod Waters benchtop Protein Characterization System combines high sensitivity and exact mass measurement. It incorporates the Waters Micromass Q-Tof micro, Waters nanoacquity UPLC System, MassLynx 4.0 and ProteinLynx Global SERVER software. Peptide sequence from BSA NanoLockSpray corrected data (MassSeq 10ppm tolerance) Nominal mass data (MassSeq 100ppm) FKDLGEEHFK FKDLGEEHFK FKDLGEE RK K LCVLHEKTPVSEK LCVLHEKTPVSEK LCVL RLLLRRLD CCTESLVNR CCTESLVNR CCTESL KKL KVPQVSTPTLVEVSR KVPQVSTPTLVEVSR KVPQVSTPTLVEVSR LVNELTEFAK LVNELTEFAK LVNEL HTLPK GFQNALLVR (FG)QNALLVR No result YLYELAR YLYELAR YLYELAR De novo sequencing of peptides is significantly enhanced through the specificity of exact mass MS/MS. The table demonstrates how greater confidence in sequence assignment is achieved with improved mass accuracy. Results are shown for a digest of Bovine Serum Albumin using ProteinLynx Global SERVER software. Correctly assigned residues Incorrectly assigned residues Ambiguous assignment Novel and homologous sequences with de novo sequencing (MassSeq) and the on-board BLAST tool In addition, you can completely automate your analysis from databank searching and AutoMod to de novo sequencing and BLAST analyses with the WorkFlow Designer tool

6 Protein expression profiling and biomarker discovery Proteomics is increasingly focused on quantitative studies, allowing a greater understanding of biological systems. This requires identification from complex mixtures potentially containing many hundreds to thousands of proteins and quantitatively comparing them over a wide dynamic range and across samples to controls. Until now, these challenging tasks have limited the full potential of proteomics. The Waters Protein Expression System has been specifically developed to address the analytical challenges inherent in quantitative proteomics including: Biomarker discovery Global protein profiling Protein expression analysis Molecular diagnostics R&D Component identification in pull-down experiments Multiplexed qualitative and quantitative proteomics The Waters Protein Expression System is a sophisticated system solution combining nanoacquity UPLC, the Q-Tof Premier, Expression Informatics, optimized chemistries, standard operating procedures and comprehensive training. Multiple global expression datasets can be normalized and compared with a control. Global expression datasets can be mined post-acquisition for qualitative/ quantitative features (e.g. biomarkers) using Protein Expression System Informatics Tools. Utilizing a unique analytical strategy, the Protein Expression System provides enhanced protein identification and relative quantitation simultaneously from LC/MS data, without the use of isotope labeling. The key benefits include: Improved sampling efficiency Using a proprietary acquisition function of the Q-Tof Premier, the system allows the parallel fragmentation of co-eluting peptides by LC/MS. Accurate quantitation without isotope labeling Exact mass chromatograms are generated for every peptide, enabling outstanding quantitative statistics for every protein. Enhanced protein identification The parallel nature of the data acquisition enables a significant increase in sequence coverage and therefore, the confidence of each identified protein. Complete data mining Protein Expression Informatics enables all peptides in global expression datasets to be qualitatively and quantitatively compared across large sample sets. Qualitative and quantitative protein expression can be visualized in Waters Protein Expression Informatics. This plot displays the EMRT (Exact Mass Retention Time) signatures from 3 peptides of a well-known biomarker, C-reactive protein, in Human Serum (shown in blue). This indicates up-regulation of the protein to approximately 50-fold, in response to infection or tissue damage.

7 Characterizing protein modifications The detection, mapping and characterization of posttranslational modifications (PTMs), (e.g. phosphorylation and glycosylation), is critical to elucidating protein function. Specifically designed for successful analysis of the most complex PTMs and peptide mixtures, the Waters Protein Modification System employs the finest range of innovative technologies available today. The system offers life science researchers significant benefits including: Unmatched resolution Ideal for peptide separations, Waters nanoacquity UPLC System offers unmatched reproducibility and resolution. Highest sensitivity Utilizing T-Wave technology, the Enhanced Duty Cycle of the Q-Tof Premier provides a near 100% efficiency in detecting diagnostic ions with precursor ion discovery experiments, enabling more low abundance PTMs to be detected and characterized. Waters Protein Modifications System incorporates the Waters nanoacquity UPLC system, the Q-Tof Premier, MassLynx and ProteinLynx Global SERVER software. Analysis of complex mixtures Programmable Dynamic Range Enhancement on the Q-Tof Premier enables exact mass measurement and linear quantitation over a concentration range of at least Targeted analysis Wizard-driven, patented Precursor Ion Discovery (PID) enables you to target your analyses specifically for modified peptides across a wide mass range by monitoring for neutral loss from phosphopeptides or product ions from glycopeptides. Maximized Proteome Coverage The Q-Tof Premier employs an exchangeable vacuum MALDI source. MALDI MS/MS on the Q-Tof Premier is particularly well suited to the sensitive, detailed analysis of phosphorylated and glycosylated peptides. Overlaid chromatograms from six consecutive separations, demonstrating retention time reproducibility >0.1minutes. Data of a yeast enolase digest (500 fmol), separated using the nanoaquity UPLC system (250 nl/min) and a 75 μm i.d. nanoacquity column packed with Symmetry C 18. LOCKSPRAY BAFFLE LOCK MASS REFERENCE SPRAY T-WAVE ION GUIDE RESOLVING QUADRUPOLE DRE LENS TRANSFER OPTICS T-WAVE COLLISION CELL ANALYTE SPRAY PUSHER DETECTOR ISOLATION VALVE AND REMOVABLE SAMPLE CONE OIL-FREE SCROLL PUMP AIR-COOLED TURBOMOLECULAR PUMPS Q-Tof Premier in MS/MS and W-Optics (17,500 FWHM) mode. 4 REFLECTRON Sequencing of a singly protonated phosphopeptide ion from PrkC by MALDI-QTOF-MS/MS. The quadruply phosphorylated peptide displays a near complete series of y ions, which confirm the sites of all four modified threonine residues. (A. Stensballe and O. N. Jensen. Rapid Commun. Mass Spectrom. 18. pp )

8 Protein separations Gaining insight into protein structure and function in complex proteomics samples requires significant reduction in sample complexity in order to achieve improved dynamic range. 2D gel electrophoresis provides high resolving power, but isn t capable of meeting all the challenges involved, particularly in the isolation of proteins in ph and molecular weight extremes. In addition, 2D gels lack the dynamic range and day-to-day reproducibility required for robust analysis of complex protein and peptide mixtures. Waters now offers an automated, highly reproducible, complementary approach to 2D gels with the introduction of the Alliance Bioseparations Systems, including high-resolution BioSuite and Symmetry300 columns. The Alliance Bioseparations System is a complete, integrated platform for 1D and 2D LC analysis of proteins, peptides and other biomolecules. Robust 1D and 2D LC analysis The Alliance 2796 Bioseparations Module is a high pressure, high-resolution HPLC system for the fractionation of complex biological mixtures. A titanium and PEEK flow path offers robust operation under harsh salt and ph conditions. With MassLynx software, you have easy control of one or two-dimensional separations and automated fraction collection. This high-pressure system is ideal for: 1D LC separation of complex protein/peptide mixtures Off-line 2D LC protein purification with fraction collection Peptide mapping 2-Dimensional HPLC (IEX/RP) analysis of an E.coli cytosolic fraction.

9 Protein interactions The characterization of protein interactions provides detailed insight into the mechanism of biological pathways and disease processes. Protein complexes and protein-protein or proteinligand interactions are readily analyzed with ESI-MS on the LCT Premier orthogonal acceleration time-of-flight mass spectrometer. Determine intact protein masses and study interactions with Waters LCT Premier Mass Spectrometer. The LCT Premier is an ideal solution offering: 10x improved sensitivity over the classic LCT Optimized ZSpray ESI source for effective transmission of protein complexes to the oa-tof analyzer LockSpray for routine exact mass measurement of intact proteins The LCT Premier mass spectrometer with ZSpray, MassLynx 4.0 and MaxEnt 1 software is an optimized platform for accurate mass determination of intact proteins and analysis of non-covalent protein interactions. W-Optics (10,000 FWHM resolution) for improved resolving power Dynamic Range Enhancement (linear dynamic range of ~10 4 ) for analyzing complex mixtures MassLynx 4.0 software for easy control MaxEnt 1 software for mass determination of proteins from multiple-charged ESI spectra The LCT Premier ZSpray source. (a) (b) Protein Analyzed Expected Molecular Mean Measured Molecular Error mda/ppm Weight (Da) Weight ±SD (Da) PttXET16A E85A ± / -0.3 HexNAc 2 Hex 8 ±1.0 ppm PttXET16A E85A ± / -2.6 HexNAc 2 Hex 9 ±1.3 ppm PttXET16A E85A ± / -0.1 HexNAc 2 Hex 10 ±0.7 ppm PttXET16A E85A ± / 11.4 HexNAc 2 Hex 11 ±3.9 ppm Accurate, intact mass determination of glycosylated isoforms of Xyloglucan endotransglycosylase mutant protein from P. pastoris. (a) ESI-MS spectrum (b) Mass determination. (Sample courtesy of Prof. Harry Brummer, AlbaNova University Centre, Stockholm, Sweden) Analysis of a high mass protein complex by ESI MS. Analysis of the Proteasome 20S multimer on the LCT Premier reveals a multiply-charged series from m/z 12,000 and 13,500 representing a complex of 685 kda molecular weight, and m/z 14,000 to 15,500 representing a complex of above 1 MDa.

10 Chemistry products for proteomics and the life sciences Robotic sample preparation systems, specialized reagents, kits and cleanup technologies together with Waters MassPREP target plates for both large and small molecule applications A range of NanoEase Capillary, Nano and Trapping columns for LC and 2D LC separations of biological components The nanoacquity UPLC columns are designed specifically for the nanoacquity UPLC System Innovative, high quality MassPREP reference standards and purified matrices to calibrate and troubleshoot chromatographic and mass spectrometry systems, simplifying system validation and ensuring reproducibility of results The RapiGest SF Reagent dramatically improves protein digestions in terms of speed and peptide recovery with no interference in HPLC, UPLC, LC/MS or MALDI/MS applications Sales Offices: Austria Australia Belgium and Luxembourg Brazil Canada X2205 Czech Republic Denmark Finland France Germany Hong Kong Hungary India Ireland Worldwide services and support Analytical Instrumentation and Software Services include Total Assurance Plans and Warranties that extend and enhance the original warranty you receive when you buy a Waters product. These plans minimize the level of insurance investment and deliver the value you need to avoid costly and time-consuming system downtime. Connections Compliance Services provide you with timely and cost-efficient solutions for your regulatory compliance challenges. You can use Waters Compliance Services to verify proper equipment operation for CGMP/GLP compliance, significantly reducing operating costs. Connections University is the center of our Educational Services, providing extensive HPLC and MS training and education at your site, at our corporate headquarters or at our local offices around the world. Representatives of Waters Global Customer Assurance Organization, trained and certified in all Waters products and current in LC and MS applications, are available in person, on the phone, via FAX or at to answer questions and provide you with service, support and information. Italy Japan Korea Mexico The Netherlands Norway People s Republic of China Poland Puerto Rico Russia/CIS Singapore Spain Sweden Switzerland Taiwan UK US WATERS CORPORATION 34 Maple St. Milford, MA U.S.A. T: F: Waters, Micromass, Q-Tof, Alliance, BioSuite, LCT, LCT Premier, Q-Tof Premier, RapiGest, MassPREP, PROtarget, nanoacquity UPLC, Ultra Performance LC, UPLC, NanoEase, Symmetry, Symmetry300, MALDI micro MX, PSD MX, Q-Tof micro, MassLynx, ProteinLynx, LockSpray, NanoLockSpray, Data Directed Analysis, DDA, MaxEnt, AutoMod, MassSeq, T-Wave, W-Optics, ZSpray and Connections are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation Printed in the U.S.A. March EN LW-CV