REDOX PROTEOMICS Roman Zubarev Roman.Zubarev@ki.se Physiological Chemistry I, Department for Medical Biochemistry & Biophysics, Karolinska Institutet, Stockholm
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
Omics cascade Action
RedOx Proteomics Reversible modifications Cys, Met, Sec (selenocysteine) Irreversible modifications Trp, Tyr, Arg, Lys,
214,000 Cys residues in the proteome
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
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
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 1.00782510 99.9852% 1.00794 2 H (D) 2.01410222 0.0148% 12 C 12.0(0) 98.892% 12.011 13 C 13.0033544 1.108% 14 N 14.00307439 99.635% 14.00674 15 N 15.0001077 0.365% 16 O 15.99491502 99.759% 15.9994 17 O 16.9991329 0.037% 18 O 17.99916002 0.204% 31 P 30.9737647 100% 30.9737647 32 S 31.9720737 95.0% 32.066 33 S 32.9714619 0.76% 34 S 33.9678646 4.22% 36 S 35.967090 0.014%
Molecular mass is the isotopic distribution! Mass defect nominal mass C=12 N=14 monoisotopic mass C=12.000(0) N=14.00307439 100 90 80 70 60 50 40 30 20 10 0 3 480 Isotopic shift 3 485 average mass C=12.011 N=14.03 C 153 H 224 N 42 O 50 3 490 3450 3455 3460 Molecular mass, Da
Molecular mass is the isotopic distribution! Selenium
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] 19+ 989.4 989.6 989.8 990 m/z 990.2 990.4 990.6 988 990 m/z 992 994 996 MS/MS ECD, CAD, CID,HCD, IRMPD, UVPD Protein ID All modifications 500 750 1000 1250 m/z 1500 1750 200 https://prosightptm.scs.uiuc.edu/
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] 3+ 0 50 100 150 Time, min Survey MS scan 628.5 629 629.5 m/z 630 630.5 631 MS/MS CAD/HCD ECD/ETD 600 700 800 m/z 900 1000 1100 120 500 750 1000 1250 m/z 1500 1750 200 Thx: Yu. Tsybin https://prosightptm.scs.uiuc.edu/ Protein ID Some modifications
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: 817.44 Fragment masses 232.17 346.22 388.20 444.28 484.33 511.37 555.40 623.45 666.44 712.52 Your Peptide/ protein is this: Score = 77
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
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%
ESI The Nobel Prize John Fenn 1917-2010 Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 246 (1989) 64-71. 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 600.. 800 V Spraying rate 0.01.. 0.1 µ l/min Spraying current in Chemistry 2002 + + na + ESI produces multiply-charged ions, [M + nh] n+ and [M - nh] n-
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. 1997:36,5554-9. denatured Charging of proteins is apprx. proportional to surface area
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 - 2004 UV Photodissociation Metastable-Induced Dissociation of Ions 2005 x y Infrared 1960s, 1995 Multiphoton Dissociation (IRMPD) z Electron Transfer 2004 Dissociation (ETD)
Near-complete sequencing of 15 kda protein Kjeldsen, F.; Zubarev, R.A. JACS, 2003, 125, 6628.
REDOX Proteomics Reduce S-S Leichert et al., PNAS 2008
IodoTMT Cys-labeling reagents TMT-10: Enables Multiplexing
RedOx Proteomics Proteins reduced by the drug Proteins oxidized by the drug
Proteome-wide Met oxidation analysis Ghesquiére et al., Mol Cell Proteomics 2011
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!