prime Innovation with Integrity The multidimensional path to the Proteome Mass Spectrometry

Transcription

1 prime The multidimensional path to the Proteome Innovation with Integrity Mass Spectrometry

2 Reach for the Full Potential of Proteomics. Open Your Eyes to PRIME The Proteome is far more complex than was ever expected at the genesis of the Proteomics revolution. Dynamics in time, space and concentration as well as variability due to modifications and mutations require novel and complementary approaches to generate useful, reliable and complete information. There is no one size fits all in unraveling the Proteome. PRIME delivers a multidimensional toolbox to unlock the complexity. Bruker s PRIME solution (Protemics through Integrated MALDI and ESI) leads the way to integrated approaches for modern proteomics and harnesses the strengths of a range of mass spectrometry technologies as well as bioinformatics without compromises. This results in enhanced protein identification and data rich interpretation about the proteome. Dependence on a single technology restricts opportunities to investigate the proteome from all directions and reduces the overall coverage and interpretation needed to truly elucidate complexities in biological systems. Within Prime, Bottomup and top-down analyses, as well as indepth protein characterization are merged to complete the proteome s puzzle. High confidence is provided by gold standard data quality and the generated information can be linked to existing biological knowledge. Novel approaches like MALDI Molecular histology are fully embedded and offer completely new approaches for complex proteomics projects. PRIME moves you, without technology compromises, to the highest level of proteome analysis. Bottomup Topdown Identify Quantify Knowledge Retrieval Reporting PTM Analysis MALDI Imaging Characterize Archiving ESI & MALDI data ProteinScape Bioinformatics

3 Bottom-up Protein Identification Bruker s Next-generation MS technologies enable greater proteome coverage in Bottom-up proteome analyses Any Bruker ESI or MALDI mass spectrometer is able to provide a new level of performance for complex proteomic samples, i.e. to identify > 2,500 proteins (single LC-MS/MS run with a False Discovery Rate FDR < 1%). PRIME: Taking advantage of ESI / MALDI complementarity ESI and MALDI represent complementary ionizations. In the shot-gun analysis of the human cell line, a 20% higher number of confidently identified proteins was achieved by combining ESI and MALDI data. This just takes three mouse clicks in the ProteinScape software. m/z 1400 maxis impact: ,017 protein ID 1% FDR Trypsin digested human colon carcinoma cell line 34,583 spectrum matches Time [min] ultraflextreme: 2,814 protein IDs 11,960 spectrum matches amazon speed: 2,544 proteinid s 22,143 spectrum matches Compiled ESI+MALDI results (ProteinScape software) 3,526 non-redundant protein IDs

4 Top-down Protein Characterization Top-down protein analysis complements Bottom-up data and generates most valuable knowledge about full protein details like sequence, splicing or proteolytic processing variants and post-translational modifications (PTM). MALDI-Top-down Sequencing (TDS) spectra even have true de-novo protein sequencing quality. PRIME combines various instruments to get the full picture. De-novo MALDI Top-down sequencing of an intact 13.6 kda Camelid antibody yields full sequence coverage. ultraflextreme Validation of the derived Camelid MoAB sequence by accurate molecular weight determination and high fidelity isotope patterns Calc. MW: Da Meas. MW: Da Mass error: 60 ppb Mass resolution > 40,000 maxis 4G maxis 4G data confirmed the expected sequence within a mass error of 60 ppb. The perfect match between the experimental (black) and expected isotope pattern (green) excluded the possibilitiy of partial deamidation or incomplete reduction of disulfide groups. Resemann A et al., Anal Chem. 2010;82(8):

5 The maxis UHR-QTOF is the ideal platform for the analysis of intact proteins, regardless of their size. Small and medium sized proteins can be measured with full isotopic resolution. Analysis of larger proteins, such as antibodies, reveals fine structures in the Maximum Entropy deconvoluted spectra, which can be attributed to glycosylation of the antibody (see Figure below). 36kDa maxis 4G LC/MS intact protein analysis of an e. coli cell lysate. Mass resolution at the protein mass 36 kda is R= 65,000. Intact antibody analysis revealing the glycosylation structure The versatility of amazon ion trap instruments makes them ideally suited to intact protein sequence analysis of small-tomedium sized proteins. By utilizing the SYNL LGFLQ RSSNF QCQKL LWQLN GRLEY CLKDR MNFDI PEEIK QLQQF QKEDA ALTIY MLQN IFAIF RQDSS STGWN ETIVE NLLAN VYHQI NHLKT VLEEK LEKED FTRGK LMSSL LKRY YGRIL HYLKA KEYSH CAWTI VRVEI LRNFY FINRL TGYLR N powerful combination of Electron Transfer Dissociation (ETD) and Proton Transfer Dissociation (PTR), fragments with up to 8+ charges can be resolved and subsequently deconvoluted at the amazons s excellent mass resolution of 30, MSYNL LGFLQ RSSNF QCQKL LWQLN GRLEY CLKDR MNFDI PEEIK QLQQF QKEDA ALTIY EMLQN IFAIF RQDSS STGWN ETIVE NLLAN VYHQI NHLKT VLEEK LEKED FTRGK LMSSL HLKRY YGRIL HYLKA KEYSH CAWTI VRVEI LRNFY FINRL TGYLR N ETD/PTR spectrum of intact β-interferon (MW ca kda) after monoisotopic peak picking and deconvolution. The N- and C-terminal sequences are fully confirmed and the read-out extends up to the disulfide crosslink at C31 - C141. amazon speed

6 Post-translational Modifications The function of many proteins is modulated by reversible PTMs. Labile PTM s like phosphorylations or glycosylations are cleaved off during conventional CID impeding the unambiguous assignment of the modification site. Since it preserves the PTM bond to the peptide backbone, ETD has become the method of choice for the analysis of labile PTMs. Its specific fragmentation pattern makes it an instrument of choice for the detection of other modifications like iso Asp which can t be adressed with CID. With its most sensitive, robust and reliable ETD technology the amazon speed ETD has proven to be the unrivalled performance leader on the market for these applications. No CID automs/ms Neutral Loss Detected? CID Yes ETD of modified peptide amazon speed s datadependent PTMScan TM for neutral loss triggered ETD (e.g., 98 Da for phosphorylation). CID ETD ETD spectrum of the peptide EELMSpSDLEETAGSTSLPK (nucleolar protein 5A) comprising 6 potential phosphorylation sites (bold). In contrast to CID, ETD allows for the unambiguous assignment of the pser at position 6. ETD amazon speed Asp containing peptide isoasp containing peptide z-57 c m/z c z c+58 z-57 ETD MS/MS of an isoasp containning peptide. The ETD fragments c+58 and z-57 allow the differentiation from aspartic acid.

7 Post-translational Modifications: Glycosylation Glycoproteomics finally unraveled - fully integrated workflow in ProteinScape Protein glycosylation is the most challenging PTM in proteomics studies, due to its complexity and lack of dedicated post-processing tools. Bruker`s ProteinScape software now enables the detection, classification and identification of glycopeptides. The peptide moiety is identified by the usual protein DB search. In addition, ProteinScape s novel proprietary GlycoQuest TM glycan DB search engine retrieves the glycan structure. The result is a complete glycopeptide elucidation from tryptic protein digests without the need for any glycan modification chemistry. GlycoQuest DB search Glycans Identified Classify Completely revealed glycopeptide structures classified spectra of glycopeptides 10,000s of MS/MS spectra Mascot DB search Peptides Identified Classifier pattern The ultraflextreme MALDI-TOF/TOF generates characteristic MS/MS fragmentation patterns which enables the classification of N-linked glycopeptides and the assignment of their complete structure in ProteinScape. (Wuhrer, J. Chromatogr. B, 2007, 849: ) amazon speed ultraflextreme Released glycans can be easily identified with the amazon ion trap. Glycan MS/MS spectra are interpreted by GlycoQuest TM glycan DB search.

8 MALDI Molecular Tissue Imaging The spatially resolved molecular view into biology and disease MALDI Imaging has emerged as a fully loaded proteomic tool. Determination of protein expression directly on cancer tissue, e.g., can reveal significant markers and lead to new insight into biological processes. Bruker s PRIME solution provides biomarker discovery and identification in an integrated Topdown proteomic strategy. Starting with tissue screening by MALDI, statistical tools pull out differential proteins. These are identified by ETD/PTR approaches in the amazon ion trap. The workflow below shows the study on HER2 + and HER2 - breast cancer tissue biopsies, which revealed CRIP 1 as a putative biomarker. HER 2+ Breast Cancer Tissue: Discovery of a putative biomarker in MALDI Imaging CRIP1 MALDI HER2 IHC CRIP1 CRIP1 HPLC fractionation of intact proteins Biomarker fractions analyzed by Top-Down ETD/PTR. Identification of the biomarker Take advantage of Bruker s leading technologies ImagePrep sample preparation The MALDI Molecular Imager encompasses the whole MALDI Imaging workflow comprising: MALDI with 1 khz laser smartbeam II technology Dedicated software for data acquisition and data visualization Sophisticated statistical analysis for expression profiling Protein markers studies for clinical endpoints by MALDI-Imaging: Rauser S, et al. Classification of HER2 receptor status in breast cancer tissues by MALDI imaging mass spectrometry. J Proteome Res ;9(4): Cazares LH, et al. Imaging mass spectrometry of a specific fragment of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 2 discriminates cancer from uninvolved prostate tissue. Clin Cancer Res. 2009;15(17):

9 Bioinformatics: ProteinScape The bioinformatics tool pulling together all your data to unravel the complexity of the proteome State-of-the-art MS instruments acquire large amounts of data at tremendous speed. To keep pace in data analysis, advanced software tools are required. In PRIME, ProteinScape, Bruker s market-leading proteomics database system, enables data analysis at an unsurpassed level of efficiency for protein identification, quantitation and PTM characterization, as well as detailed reporting in accordance with publication guidelines. ProteinScape s intuitive user interface provides access to any detail of proteomic datasets (separation, protein and peptide IDs, MS and MS/MS spectra) by a single mouse click. Projects can be structured to precisely reflect the analytical workflows applied in the lab. Gene ontology (GO) data can be imported for individual or groups of identified proteins. Data from different MS platforms can be merged resulting in extremely confident, high quality proteomics information.

10 Bioinformatics: ProteinScape Turning proteomics data into biological information Link to Web Knowledge ProteinScape connects to web resources for higher order information such as 3D structures of identified proteins. Identified peptides and modifications can be mapped to the protein structure (in the figure below phosphorylation in red) to facilitate the elucidation of the structure-function relationship in biological processes. Queries The data of entire projects can be queried for specific protein or peptide features such as N-glycosylation sites or other PTMs. As shown on the right, individual query results can be compared in Venn diagrams. Such comparative queries facilitate fast and convenient method optimization, e.g. for the enrichment of N-glycopeptides using different lectin stationary phases. Protocol LCA 3115 Boronic acid 3621 WGA 3160 ConA 3967 # N-glycosylated peptides Essential features of ProteinScape in the proteomics workflow Identify Quantify Characterize Integrate Queries Reporting Multiple search engines Decoy searches Non - redundant protein lists Non - and isobaric labels Label - free Combined quantitation from different fractions PTM discovery by CID, ETD, etc. Spectra classification for glycopeptides and glycans GlycoQuest DB search for glycans Merges results from multiple separations, MS and search engines Generation of Scheduled Precursor Lists (SPL) Links with GO terms and 3D structures peptide, protein, glycan, spectral features Comparative queries for optimization HUPO/PSI publication guidelines Detailed protein and glycan reports

11 Quantification with label and label-free strategies Identification and quantification of protein expression is key for the understanding of biological processes and for the discovery of biomarkers. ProteinScape offers full support for label (SILAC, itraq, ICPL, etc.) or label-free approaches in a seamless fashion. Advanced visualization and validation tools are accessible through an easy-to-use interface, allowing straightforward extraction of essential information from quantitative proteomic studies. ProteinScape supports PRIME: Interaction between identification and quantitative data from all Bruker instruments. Search results can be combined before quantification. Statistical data handling tools, e.g normalization, statistical viewers, etc. Colon tissue normal cancer Identification, structural characterization and quantification at a glance Ultimate dynamic range ultraflextreme Label-free quantification of neural progenitor cell proteins (ultraflextreme). The Volcano plot in ProteinScape shows > 2x regulated peptides (green dots). Down to low amol label-free quantitation of a UPS 1 digest spiked in a 1:2 concentration ration in 500ng of an E.coli digest. UPS 1 is a digest of 48 proteins covering 6 orders of magnitude in concentration dynamic range. Maltman DJ et al., Proteomics Jul 15

12 Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. BDAL , # PRIME Proteomics through Integrated MALDI and ESI A multi-tier approach to decipher biology and disease, and to turn proteome data into knowledge: maxis impact, maxis 4G UHR-QTOF Ultimate combination of high resolution, mass accuracy, speed and senstivity Unlimited m/z for Top-down analysis of intact proteins and non-covalent protein complexes autoflex speed, ultraflextreme MALDI-TOF/TOF Bottom-up LC-MALDI with high dynamic range and no time contraints Top-down analysis of proteins Glycopeptide classification and complete structure elucidation MALDI Molecular Imaging on tissue amazon ion trap series Ultrafast MS/MS speed for high sequence coverage in Bottom-up proteomics ETD analysis of post-translationally modified peptides Top-down ETD/PTR of intact proteins CaptiveSpray nano/cap sprayer Robust and reliable plug-and-play nano/cap-esi source ( nl/min) High transmission by focusing the ion plume into the MS orifice Advance nanolc Splitless nanouhplc system (up to 10,000 psi) for 1D and 2D separations Reliable, reproducible and robust to maximize MS utilization time ProteinScape bioinformatic suite Market-leading proteomics data analysis system Highly integrative tools for in-depth knowledge retrieval Archiving and reporting according to standard guidelines For research use only. Not for use in diagnostic procedures. Bruker Daltonik GmbH Bremen Germany Phone +49 (0) Fax +49 (0) sales@bdal.de Bruker Daltonics Inc. Billerica, MA USA Fremont, CA USA Phone +1 (978) Phone +1 (510) Fax +1 (978) Fax +1 (510) ms-sales@bdal.com ms-sales@bdal.com