Fan Liu Utrecht University, The Netherlands

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Presentation transcript:

Charting the cellular interactome by proteome-wide cross-linking mass spectrometry   Fan Liu Utrecht University, The Netherlands Netherlands Proteomics Center

Introduction of cross-linking mass spectrometry (XL-MS) XL-MS workflow Maximal distance constraints Linker length Sidechain length Distance constraint between C 11.4 Å 6.4 Å 24.2 Å 11.4 Å DSS Disuccinimidyl suberate

Introduction of cross-linking mass spectrometry (XL-MS) XL-MS workflow

Proteome-wide XL-MS Using MS-cleavable cross-linker Implementing combined fragmentation strategies Developing XlinkX v2.0 search engine

Solving the n-square problem Proteome-wide XL-MS Using MS-cleavable cross-linker Solving the n-square problem Disuccinimidyl Sulfoxide (DSSO) Kao A., et al. Mol Cell Proteomics, 2011

Solving the n-square problem Proteome-wide XL-MS Using MS-cleavable cross-linker Solving the n-square problem Disuccinimidyl Sulfoxide (DSSO) Mass Intensity MS2 AL BL AS BS Liu F., et al. Nat Methods, 2015

Solving the n-square problem Proteome-wide XL-MS Using MS-cleavable cross-linker Solving the n-square problem Liu F., et al. Nat Methods, 2015

Proteome-wide XL-MS Using MS-cleavable cross-linker Implementing combined fragmentation strategies CID-only Liu F., et al. Nat Methods, 2015

Solving the n-square problem Proteome-wide XL-MS Using MS-cleavable cross-linker Implementing combined fragmentation strategies Solving the n-square problem CID-ETD MS2 dual play Liu F., et al. Nat Methods, 2015

Solving the n-square problem Proteome-wide XL-MS Using MS-cleavable cross-linker Implementing combined fragmentation strategies Solving the n-square problem CID-ETD MS2 dual play Ion Routing Multipole C-Trap Ion Trap HPC LPC MP0 Q1 m/z Full FTMS Scan Orbitrap m/z 1st MS2 CID Scan m/z 2nd MS2 ETD Scan Full OT MS scan 1st CID MS2 on MS precursor ion in OT 2nd ETD MS2 on MS precursor ion in OT Liu F., et al. Nat Methods, 2015

Solving the n-square problem Proteome-wide XL-MS Using MS-cleavable cross-linker Implementing combined fragmentation strategies Solving the n-square problem CID-only CID-ETD MS2 dual fragmentation Liu F., et al. Nat Methods, 2015

Solving the n-square problem Proteome-wide XL-MS Using MS-cleavable cross-linker Implementing combined fragmentation strategies Solving the n-square problem CID-MS2-MS3 dual play Liu F., et al. Manuscript in preparation

Solving the n-square problem Proteome-wide XL-MS Using MS-cleavable cross-linker Implementing combined fragmentation strategies Solving the n-square problem CID-MS2-MS3 dual play Ion Routing Multipole C-Trap Ion Trap HPC LPC MP0 Q1 m/z Full FTMS Scan Orbitrap m/z 1st MS2 CID Scan m/z 2nd MS3 CID Scan m/z 2nd MS3 CID Scan m/z 2nd MS3 CID Scan m/z 2nd MS3 CID Scan Full OT MS scan 1st CID MS2 on MS precursor ion in OT 2nd mass difference dependent MS3 scans in IT Liu F., et al. Manuscript in preparation

Solving the n-square problem Proteome-wide XL-MS Using MS-cleavable cross-linker Implementing combined fragmentation strategies Solving the n-square problem CID-MS2-MS3 dual fragmentation Liu F., et al. Manuscript in preparation

Proteome-wide XL-MS Using MS-cleavable cross-linker Implementing combined fragmentation strategies Developing XlinkX v2.0 search engine Liu F., et al. Manuscript in preparation

Proteome-wide XL-MS application (E.coli) Liu F., et al. Manuscript in preparation

Proteome-wide XL-MS application (mitochondrion) Electron transfer chain supercomplexes (mitochondrion) Proposed in 1955 (Chance & Williams) Weak experimental evidence COX7A1/2 COX5A COX4i1 COX6B1 COX7B CIV CI CIII CII NDUFV1 NDUFA9 NDUFA5 NDUFA4 NDUFB10 NDUFA11 NDUFB4 NDUFB7 UQCRC1 UQCRC2 UQCRH UQCR10 SDHA

Summary Method development of proteome-wide XL-MS The use of MS-cleavable cross-linkers The implement of combined fragmentation strategies The development of XlinkX search engine (XlinkX PD nodes) Biological applications Intact organelles Whole cell lysates

Acknowledgements Utrecht University Prof. Albert Heck Philip Lössl Dr. Dirk Rijkers Harm Post Dr. Richard Scheltema National Heart, Lung, and Blood Institute Prof. Robert S. Balaban Beverley M. Dancy Thermo Fisher Scientific Rosa Viner Bernard Delanghe Torsten Ueckert Kai Fritzemeier

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