Designed for Accuracy. Innovation with Integrity. High resolution quantitative proteomics LC-MS

Transcription

1 Designed for Accuracy High resolution quantitative proteomics Innovation with Integrity LC-MS

2 Setting New Standards in Accuracy The development of mass spectrometry based proteomics approaches has dramatically improved researchers capabilities of characterizing the protein content of biological systems, while simultaneously offering information on proteins identity, abundance and modifications. Accurate and reproducible protein quantitation is key for success and Bruker s latest UHR-QTOF platforms are designed to provide the best performance in this area. The Instant Expertise acquisition methods provide easy and straightforward access to the underlying biology. Generic quantitation workflows Chromatographic separation Ionization Extracted proteins from biological samples Peptides Digestion Chromatographic separation CaptiveSpray + nanobooster

3 Accuracy = Resolution x Sensitivity Full Sensitivity Resolution (FSR) In traditional QTOF architecture, high resolution is obtained at the price of ion losses through slits or multiple reflections and the maximal sensitivity is therefore not accessible at full resoultion. The Bruker FSR architecture, in contrast, makes use of the maximum number of ions to obtain the highest resolution available without sensitivity loss. This ensures the best possible selectivity and sensitivity, at any speed. Whatever strategy is chosen for quantitative mass spectrometry based analysis of complex proteomics samples, the most important aspect is accurate determination of ratios both in single runs or large sample cohorts. Data acquisition Post processing Quantitative results proteinscape MaxQuant Perseus impact II Skyline

4 Repeatable High Performance The impact II s unique combination of sensitive, high resolution and high dynamic range spectra opens a new dimension in MS based quantitation. Both label-free quantitation workflows and quantitation based on chemical labels are very easy to implement with pre-set methods. Preservation of spectral quality in MS/MS mode guarantees optimal performance for these data dependant acquisition (DDA) approaches. Instant Expertise The impact II s default ID/Quant acquisition method ensures accurate MS based quantitation with regular spacing of MS events. The number of MS/MS events and the corresponding acquisition speed is adapted depending on parent ion intensity. Optimal MS/MS based quantitation is ensured by transfer time stepping functionality that allows high sensitivity over the entire mass range. Instant Expertise Instant Expertise for label free and metabolic labels Optimal chromatographic shape for accurate quantitation High resolution for more selectivity Instant Expertise for isobaric chemical labels Broadband MS/MS transfer for optimal reporter ion quantitation and peptide identification Instant Expertise label free and metabolic labels Instant Expertise isobaric chemical labels MS spectrum from potato root digest in an LC-MS/MS experiment showing >50,000 resolution for a selected parent ion. (Data courtesy of Dr. Hans Peter Mock, IPK Gatersleben) itraq8 MS/MS spectrum from E.coli digest spiked with four proteins at ratios 10:10:5:5:2:2:1:1. (Samples courtesy Dr. David Ashford, York University)

5 Across the Whole Range The impact II allows targeted proteomics experiments to be run in either an MS based format or at the MS/MS level based on data independent acquisition (DIA). Superior resolution and full sensitivity of MS and MS/MS spectra facilitate both approaches on the impact II. Low method development requirements Data aquisition in MS mode for targeted peptide and protein quantitation provides accurate results over a large dynamic range based on high resolution extracted ion chromatograms (Hr-XIC). This mode is applicable to both high and low complexity samples and both approaches benefit from the uncompromised resolution and sensitivity of the impact II. In DIA, the relevant mass range for proteomics is covered by larger isolation windows to obtain high resolution MS and MS/MS information of each peptide contained in the sample. Targeted HR-XIC Data Independent Acquisition (DIA) Read more: Bruker Daltonics Application Note LCMS-89 Ceruloplasmin peptide quantitation Ceruloplasmin peptide quantitation over 4 orders of magnitude (10 amol fmol/µl) in non depleted plasma.

6 Label-Free Intact Protein Analysis Peptide based quantitation is a very sensitive approach but it is blind to some essential biological events such as protein proteolysis. By combining Full Sensitivity Resolution with the True Isotopic Pattern (TIP ) capability of Bruker s UHR-QTOF systems, accurate measurement of intact proteins is readily available, even in complex mixtures. The Dissect and SNAP algorithms extract the accurate mass and intensity information to perform a direct label free approach on intact proteins. Dissect Dissect processing of an E.Coli lysate separated with a 20 min gradient recovers more than 120 coelution peaks, corresponding to more than 800 distinct proteoforms. SNAP Deconvoluted intact protein spectrum after 5.2 min. The monoisotopic mass was calculated using SNAP that takes the experimentally determined very accurate isotopic pattern (True Isotopic Pattern) into account. Statistical analysis of label free protein profiling study Volcano plot displaying quantitative results. Read more: Bruker Daltonics Poster Note PN-04

7 Label-Free Shotgun Proteomics Accuracy of the label free quantitation (ID/Quant) workflow Thanks to the combination of very stable nano electrospray (CaptiveSpray ) and advanced QTOF technology, the impact II offers robust and accurate labelfree quantitation. Since this strategy relies on every single peptide precursor ion that is detected at the MS level, the benefit of high resolution over the entire mass range directly translates into superior ratio determination. Control Digestion LC-MS/MS Peak intensity Sample Quant accuracy over a large dynamic range 500 ng of yeast digest were spiked with UPS2 protein standard (Sigma-Aldrich, P/N UPS2) in a 1:2 ratio. After injection, UPS 2 proteins were identified and quantified over 4 orders of dynamic range, despite the small ratio difference. (Data courtesy S. Beck et al, MaxPlanck Institute for Biochemistry, Martinsried, Germany) Read more: Journal of the MCP: The impact II, a very high resolution quadrupole time-of-flight instrument for deep shotgun proteomics

8 Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. BDAL , Verified by Science The impact II provides latest Bruker UHR-QTOF technology The unique combination of quantitative accuracy, dynamic range and reproducibility make the system capable of delivering quantitative data with best accuracy. CaptiveSpray nanobooster Source Zero adjustment for excellent reproducibility Acetonitril dopant for improved sensitivity Full-Sensitivity-Resolution Technology 50,000 resolving power at MS and MS/MS level without compromising sensitivity or acquisition speed Highly accurate extracted ion chromatogram (1-2 mda) Patented Dual Ion Funnel Optimized ion transmission Off axis ion inlet for improved robustness 50 Gbit/s Detection System High intra spectral dynamic range at LC-MS speed Collision Cell Improved transmission for optimal sensitivity Wide m/z transfer TOF technology has fundamental attractions, such as the extremely high scan speed and the absence of space charge, which limits the number of usable ions in all trapping instruments. TOF instruments [are] capable of making use of the majority of ions, thus promising optimal sensitivity, dynamic range and hence quantification. It appears that QTOFs are set to make a comeback in proteomics with recent examples demonstrating impressive depth of coverage of complex proteomes. For research use only. Not for use in diagnostic procedures. Bruker Daltonik GmbH Bremen Germany Phone +49 (0) Fax +49 (0) Bruker Daltonics Inc. Billerica, MA USA Phone +1 (978) Fax +1 (978) ms.sales.bdal@bruker.com -