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Optimizing Top Down Analysis of Proteins on Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer

Optimizing Top Down Analysis of Proteins on Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer

10 Aug 2015

This application note demonstrates the optimization of top down analysis of intact proteins with the goal of maximizing sequence coverage on a Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer.

Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer

Thermo Fisher Scientific

Test new limits of detection, characterization and quantitation with the latest Tribrid™ mass spectrometer. The Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer is designed to expand performance in advanced proteomics, biopharma and metabolomics applications, including quantitation using isobaric tags, low level PTM analysis, data independent acquisition (DIA), and top down proteomics. The new instrument features enhanced sensitivity resulting in improved analyte detection, characterization and quantitation, enabling scientists to perform more comprehensive sample analyses faster and with better accuracy than ever before.   Novel Orbitrap Fusion Lumos MS Features: Novel high-sensitivity API interface combines a High Capacity Transfer Tube and an Electrodynamic Ion Funnel for increased ion flux and lower limits of detection Advanced Active Beam Guide prevents neutrals and high velocity clusters from entering the resolving quadrupole Advanced Quadrupole Technology combines high selectivity and efficiency of transfer for selected ions symmetrically across the isolation window Advanced Vacuum Technology improves transmission of high molecular weight ions to the Orbitrap analyzer Novel ETD HD—high dynamic range ETD provides significantly increased fragment ion coverage Established Tribrid MS Features: Tribrid architecture — includes quadrupole mass filter, linear ion trap and Orbitrap mass analyzers Ultrahigh resolving power up to 500,000 FWHM, with isotopic fidelity up to 240,000 FWHM at m/z 200 Acquisition rates of up to 20 Hz for both Orbitrap and linear ion trap MSn analyses Full parallelization of MS and MSn analyses with intelligent ADAPT™ (All Dynamically Available Parallelizable Time) technology Synchronous Precursor Selection (SPS) for MS and MSn experiments significantly increases the number of peptides and proteins identified and improves quantitative accuracy when using isobaric mass tags Flexibility of fragmentation — CID, HCD and optional ETD and EThcD available at any stage of MSn with detection in either the Orbitrap or linear ion trap detector enables detailed structure determination of metabolites, glycans and other small molecules Universal Method provides maximal peptide identifications without method optimization for samples of unknown concentration, reducing sample and instrument time requirements for routine peptide identifi cation experiments Intuitive and flexible drag-and-drop user interface simplifies method development and enables unique and complex workflows

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Thermo Scientific™ ProSwift™ RP Protein Column

Thermo Fisher Scientific

Monolith Reversed-Phase Columns for High-Resolution Protein Separations at High Flow Rates ProSwift® reversed-phase monolith columns uniquely provide the advantages of high resolution at exceptionally high flow rates for protein separations. They deliver the outstanding resolving power of nonporous analytical media combined with faster separations than any bead based columns available. ProSwift polymer monolith columns achieve long column lifetime, exhibit reproducibility even after hundreds of runs, and have broad pH stability, providing high stringent wash capability.ProSwift polymer monolith media are uniquely suited for the separation of proteins. Each monolith is a single cylindrical polymer rod containing an uninterrupted, interconnected network of flow-through channels of a specific pore size. These large channels and the monolith’s nonporous surfaces result in fast mass transfer for high-resolution and fast protein separations. The channels also produce low backpressure, allowing the use of higher linear velocities with minimal loss of resolution.ProSwift® RP Protein Column Features: High resolution at high speed Highest operational flow rates available High throughput and improved productivity Optimal performance in a broad range of applications Excellent stability over a wide pH range of 1 to 14 Outstanding reproducibility and ruggedness High stringent wash compatible, for example, 1 M NaOH High loading capacity Column Details: Chemistry: Phenyl Length: 50, 250 mm ID: 1.0, 4.6 mm

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Optimizing Top Down Analysis of Proteins on Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer