REDOX PROTEOMICS. Roman Zubarev.

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1 REDOX PROTEOMICS Roman Zubarev Physiological Chemistry I, Department for Medical Biochemistry & Biophysics, Karolinska Institutet, Stockholm

2 What is (RedOx) Proteomics? Proteomics - large-scale analysis of proteome Proteome all polypeptides expressed in a given time in a given organ of a given organism Redox proteome - reversible and irreversible covalent modifications that link redox metabolism to biological structure and function Proteome analysis obtaining information on protein identities, sequences, modifications, structure and abundances

3 Omics cascade Action

4 RedOx Proteomics Reversible modifications Cys, Met, Sec (selenocysteine) Irreversible modifications Trp, Tyr, Arg, Lys,

5 214,000 Cys residues in the proteome

6 214,000 Cys residues in the proteome In cell & tissue proteins : 5-14% of Cys residues are oxidized, up to 40% if oxidants are added

7 Proteome dynamic range and its implications Dynamic(range(of(mass(spectrometers( 9000# 8000# 7000# 5000# 6000# 4000# Number(of(detected( proteins( 3000# 2000# 1000# Sample!"!#"!##"!###"!####"!#####"!######"!#######" Proteins,*number*of*copies*per*cell* 10#mg#########1#mg########100#µg#######10#µg########1#µg##########100#ng#####10#ng########1#ng# Sample*size* Zubarev R.A., Proteomics, 2013

8 What is Molecular Mass? Mass: M = Σm e n e, m e mass of an element n e number of atoms of this element in the molecule Isotope Mass Abundance Chemical mass 1 H % H (D) % 12 C 12.0(0) % C % 14 N % N % 16 O % O % 18 O % 31 P % S % S % 34 S % 36 S %

9 Molecular mass is the isotopic distribution! Mass defect nominal mass C=12 N=14 monoisotopic mass C=12.000(0) N= Isotopic shift average mass C= N=14.03 C 153 H 224 N 42 O Molecular mass, Da

10 Molecular mass is the isotopic distribution! Selenium

11 Top-Down Proteomics Cell lysis blood HPLC gels enzymatic cleavage PTMs protein mixture protein or large protein fragment CID protein enzymatic fragments (peptide mixture) intact cell, tissue Top-Down MS LC-MS/MS [M+19H] m/z m/z MS/MS ECD, CAD, CID,HCD, IRMPD, UVPD Protein ID All modifications m/z

12 Bottom Up Proteomics cell Lysis Blood HPLC gels enzymatic cleavage PTMs protein mixture protein or large protein fragment CID protein enzymatic fragments (peptide mixture) intact cell, tissue LC-MS/MS Bottom-Up (Shotgun) proteomics [M+3H] Time, min Survey MS scan m/z MS/MS CAD/HCD ECD/ETD m/z m/z Thx: Yu. Tsybin Protein ID Some modifications

13 Protein Identification by Tandem Mass Spectrometry Protein sequence ILNKPEDETHLEAQPTDASAQFIRNLQISNE DLSKEPSISREDLISKEQIVIRSSRQPQSQNPK LPLSILKEKHLRNATLGSEETTEHTPSDASTT EGKLMELGHKIMRNLENTVKETIKYLKSLF SHAFEVVKT Enzymatic digest Tryptic peptides EDLISK EQIVIR LPLSILK NLENTVK LMELGHK QPQSQNPK NLQISNEDLSK SLFSHAFEVVK NATLGSEETTEHTPSDASTTEGK ILNKPEDETHLEAQPTDASAQFIR Tandem Mass Spectrometry (MS/MS) Tryptic peptide NLENTVK MS/MS Fragmentation N L E N T V K Molecular mass: Fragment masses Your Peptide/ protein is this: Score = 77

14 MS-based quantitative discovery approaches SILAC TMT, itraq Label-free Cells Heavy Light Proteins H/L Heavy Light Peptides H/L H/L Biology? 0 0 Extraction Digestion Tissues, bodily fluids 0 0 N/A x x N 11 x x N-any

15 Hybrid LTQ-Orbitrap Mass Spectrometer 1. Ions are stored in the linear trap of LTQ 2. are axially ejected 3. and trapped in the C-trap and squeezed into a smaller cloud 4. then a voltage pulse across C-trap ejects ions towards the Orbitrap 5. where they are trapped and detected V Gas <1 mtorr x V -2.5 k V Central Electrode Voltage y -3.5 k V Transmission=30..50%

16 ESI The Nobel Prize John Fenn Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 246 (1989) Metal capillary + Atmospheric side - HV power supply 2..4 kv E 10 6 V/m Droplet desintegration electrons Spraying rate 1 µ l/min Spraying current Desolvation + na MS nano-electrospray Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 136 (1994) 167 Pulled glass capillary tip µm V Spraying rate µ l/min Spraying current in Chemistry na + ESI produces multiply-charged ions, [M + nh] n+ and [M - nh] n-

17 ESI of proteins native ESI mass spectra of cytochrome c in water with 3% MeOH and 0.5 mm ammonium acetate Konermann L, Douglas DJ. Biochem. 1997:36, denatured Charging of proteins is apprx. proportional to surface area

18 Backbone Fragmentation Electron 2004 Detachment Dissociation (EDD) a b Collisionally 1960s, 1990s Activated /Induced Dissociation (CAD/CID) Electron Capture 1998 Dissociation (ECD) c -CHR C(O) NH CHR UV Photodissociation Metastable-Induced Dissociation of Ions 2005 x y Infrared 1960s, 1995 Multiphoton Dissociation (IRMPD) z Electron Transfer 2004 Dissociation (ETD)

19 Near-complete sequencing of 15 kda protein Kjeldsen, F.; Zubarev, R.A. JACS, 2003, 125, 6628.

20 REDOX Proteomics Reduce S-S Leichert et al., PNAS 2008

21 IodoTMT Cys-labeling reagents TMT-10: Enables Multiplexing

22 RedOx Proteomics Proteins reduced by the drug Proteins oxidized by the drug

23 Proteome-wide Met oxidation analysis Ghesquiére et al., Mol Cell Proteomics 2011

24 RedOx Proteomics Conventional proteomics probes the abundance changes of thousands of proteins in the sample Redox proteomics can determine shifts in the oxidation state of hundreds of proteins All measurements are quantitative. Statistical significance can only be achieved through replicate analyses What happens in vivo happens also in vitro. Proper controls are needed! Validation of all important findings is required. Validation has to be done by a different method than discovery. G o o d L u c k!