New Instruments and Services

Transcription

1 New Instruments and Services Liwen Zhang Mass Spectrometry and Proteomics Facility The Ohio State University Summer Workshop 2016 Thermo Orbitrap Fusion fusion

2 Thermo Orbitrap Fusion

3 Thermo Orbitrap Fusion Combining the best of quadrupole, Orbitrap, and ion trap mass analysis in a revolutionary Tribrid architecture, the Orbitrap Fusion Tribrid mass spectrometer features: Resolution up to 450,000 to remove spectral interferences CID/HCD/ETD detected by the Ion Trap or Orbitrap at any level of MSn for maximum experimental flexibility Dynamic Scan Management architecture lets every user easily get the maximum information from every sample Quick polarity switch between positive and negative mode. Bruker SolariX XR 15T FT ICR

4 Bruker SolariX XR 15T FT ICR The ParaCell is the new core technology for solarix XR, providing uncommon broadband ion stability resulting in resolution far superior to other detection schemes. Ultimate mass resolving power in excess of RP = 10,000,000 FTICR technology providing unrivaled mass accuracy Apollo II Ion Funnel Electrospray Source enabling high ion transmission efficiency over a broad mass range Hybrid Qh Front End supporting CASI for enrichment of low abundant species Optimized ion optics for sensitivity and mass range coverage Proprietary ParaCell ICR cell with patented SIDEKICK ion accumulation system for high detection efficiency. Nanobay E Acquisition electronics enabling data streaming for increased broad band resolution Wide range of fragmentation techniques including CID and ECD Dual ionization source: MALDI source with Smartbeam II laser and ESI source

5 Thermo Q Exactive Plus Thermo Q Exactive Plus This benchtop LC MS/MS system combines quadruple precursor ion selection with high resolution, accurate mass (HRAM) Orbitrap detection to deliver exceptional performance and versatility Resolving power up to 140,000 FWHM; Fast HRAM Orbitrap technique (High mass accuracy <1ppm) eliminates interferences for high confidence structural identification and large scaleprofiling of metabolomics/lipidomics/proteomics samples Fast scanning at 12hz and spectral multiplexing suited to UHPLC applications Rapid polarity switching in both MS and MS/MS modes maximizes structural detection and identification from a single injection In source CID and HCD; HCD yields superior fragmentation and higher quality MS/MS spectra to improve identifications Extended mass range to 6,000m/z enhances detection of singly charged small molecules and biomolecules Great dynamic range (>4 orders of magnitude), along with femtogram level sensitivity, allow detection of trace level and high abundance compounds in the same scan High spectral quality and a wide dynamic range make it possible to identify and quantify proteins other instruments cannot find. Quan/qual capability make it easier to transition from proteomic discovery experiments to targeted protein quantification

6 Application of Orbitrap Fusion PROTEINS Bottom-up Proteomics Targeted Quantitation Top-Down Proteomics Intact Protein Characterization Post Translational Modification Analysis: Glycosylation Phosphorylation Relative Quantitation: SILAC TMT Label-Free GLYCANS Glycan Identification & Characterization 2-AA Quantitation Intact Glycoprotein Profiling PTM Glycosylation Application of 15T FT ICR High End Proteomics Studies (Top-down and Bottom-up workflows) PTM Investigation Accurate de novo sequencing Molecular Imaging of Tissue Distribution of Drugs, Metabolites, and Biomarkers Petroleum Product Analysis Complex Environmental Sample Analysis Metabolomics Research Application of Q Exactive Plus Targeted Proteomics Studies Quantitative Proteomics PTM Investigation with High Confidence Quantitative Metabolomics Metabolomics Research New Services: 2D LC/MSMS for Protein Identification and PTM Study Targeted Proteomics Quantitation Top-down Proteomics MADLI Tissue Imaging

7 Time (min) Time (min) High ph Fractionation with Low ph Separation Waters Mclass 2D System Dionex D System $60/fraction on the orbitrap XL $75/fraction on the orbitrap Fusion Slide courtesy of Nilini S. Ranbaduge Multi-dimensional Separation ph 10.0 ph 2.6 F1 $60/fraction on the orbitrap XL $75/fraction on the orbitrap Fusion RT: F2 Relative Abundance Time (min) Fractionation (F) Time (min) Gradient separation F15 14 Slide courtesy of Nilini S. Ranbaduge

8 Sequest HT (D2) Human HEK293 Cell 2D LC/MSMS 4ug Loading; 3 replicates Sequest HT (D2) Sequest HT (E2) Sequest HT (E2) Sequest HT (F2) Sequest HT (F2) 2577 Total Label Description 3537 Sequest HT (D2) Identified by: Sequest HT (D2) 3488 Sequest HT (E2) Identified by: Sequest HT (E2) 3497 Sequest HT (F2) Identified by: Sequest HT (F2) 3127 Sequest HT (D2)/(E2) Identified by: Sequest HT (D2)/ (E2) 3113 Sequest HT (E2)/(F2) Identified by: Sequest HT (E2)/ (F2) 3202Sequest HT (D2)/(F2) Identified by: Sequest HT (D2)/(F2) 2975 Sequest HT (D2)/(E2)/(F2) Identified by: Sequest HT (D2)/(E2)/(F2) 4055 Total Label Description Sequest HT (D2) Identified by: Sequest HT (D2) Sequest HT (E2) Identified by: Sequest HT (E2) Sequest HT (F2) Identified by: Sequest HT (F2) Sequest HT (D2)/(E2) Identified by: Sequest HT (D2)/ (E2) Sequest HT (E2)/(F2) Identified by: Sequest HT (E2)/ (F2) Sequest HT (D2)/(F2) Identified by: Sequest HT (D2)/(F2) Sequest HT (D2)/(E2)/(F2) Identified by: Sequest HT (D2)/(E2)/(F2) 33958

9 Top down v.s. Bottom up Proteomics Circulation: Cardiovascular Genetics December 2011 vol. 4 no Trends in Biochemical Science; Volume 35, Issue 9, September 2010, Pages Protein Identification by Top Down MS/MS Parents ions ( , 15+) Ac + 2Me + 2Ac + 2Me Histone H4 +1 Ac + 4Ac + 5Ac m/z ECD MS/MS c13+42 z64 c z50 z42 C24+70 c15+42 c c z65 z34 c c7+42 c c59+70 c c c26+70 c12+42 c52+70 c37+70 c z35+14 c c17+84 z12 c c40+70 c26+70 c z35+56 c12+42 c18+84 c z20 c C c54+70 c z48 z9 z32 z49+14 z49 z66 z25+16 z75 z76 z67 z51 z42+14 z59 z68 z43 z78 z62 z61 z79 z m/z m/z

10 Protein Identification by Top Down MS/MS Me Me Ac Ac Ac Ac Ac N-AGRGK 5 GGK 8 GLGK 12 GGAK 16 RHRK 20 VLRDNIQGIT Ac Ac Ac H4 Known Modification Modification Detected $500 on Fusion or ICR MALDI Imaging Image from 2014 ASMS Workshop

11 MALDI Imaging $750/Sample Trends in Biochemical Science; Volume 29, Issue 3, March 2011, Pages

12 Problem Interference from Matrix Metabolites not Stable Solutions: High Mass Accuracy (FT ICR) Changing Matrix Derivatization Using Product Ions for Scanning $750/Sample on 15T ICR Biology 2015, 4(1), Laser Capture Microdissection Proteomics Relative Abundance Time (min)