Top-Down Proteomics Mass Spectrometry

University of Florence, March 19, 2010

“Bottom-Up” of proteins : Before MS, slash, bash, smash into peptides.

2010: MS Proteomics >90% bottom-up

Fred W. McLafferty, Cornell University Top-Down versus Bottom-Up Protein Characterization by Tandem High-Resolution Mass Spectrometry

Kelleher, N. L, Lin, H. Y, Valaskovic, G. A, Aaserud, D. J, Fridriksson, E. K, McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806-812 Carbonic Anhydrase

Calculated Mr = 29023.7-17 2831.7 -1 2960.6 -1

7601.5 -4 15191 -2 15319.2 -10 6103.5 -3 7247.3, 7146.3, 7045.2 -4

4614.4 -2 10704.9 -8 4950.9 -2

20270.2 -12 8754.1 -5 20972.6 -12

21422.8 -13 4614.4 -2 15319.2 -10 b135 -b134, 128.01 : Q, -0.05 7601.5 -4 10704.9 -8 4950.9 -2 Da

29024.3 -17 6103.5 -3 29024.2 -17 y63 -y62, 101.04:T, -0.01 7601.5 -4 y62 -y61, 101.11: T, +0.06 29024.3 -17 Cornell never has BUT- done Bottom-Up Top-Down began before McLafferty J. J. Thompson, 1913 MS of Linear Molecules (1958) Mycoserosic acid (1 µg) from tubercle bacilli Showing four methyl branch positions

Rearrangement

13 28 R. Ryhage, E. Stenhagen J. Lipid Res. 1960, 1, 361. 1 microgram! 14 28 14 28

Top-Down Units: CH2, 14 Da; CH(CH3), 28 Da Problem: MS of Nonlinear Molecules

Clivonine

Hippeastrine

Solution #1: Study Klaus Biemann’s papers Not a Linear Molecule? Rearrangement? Databases of Electron Ionization Mass Spectra Wiley 1969 Registry of Mass Spectral Data, 6.8K spectra Stenhagen, Abrahamsson, McLafferty, Editors Wiley 2009 Registry, 9th Edition, 660K spectra NIST 2008 Edition, 220K spectra Wiley 2009 Registry with NIST, 9th Edition, 796K spectra 746K searchable chemical structures 667K different chemical compounds Probability Based Matching: 796K in <1 s Electrospray Ionization - John Fenn

1-3 kV

ESI is Really Gentle - BUT Is the native conformation retained? Molecular Weight, Specificity Bovine Casein in Mozzarella Cheese Legal limit 5%, used “Tasters” 1989 Electrospray Ionization MS, 1989

3 isoforms Detect 1% Bovine!

Amount of data ~ “peak capacity” = Width (m/z range) x Resolving power Fourier-Transform Ion Cyclotron Resonance EquippedFragmentation with MassMultiple Spectrometer Techniques MS/MS Techniques in FTMS Comisarow and Marshall Multiple MS/MS techniques Orbitrap, no magnet, ECD “Middle-Down” MS Electron Capture Dissociation (electron2 Electron filament andGuns emitter) 1. ECD 2. Hot e, Activation Nanospray Superconducting ESI source IRMPD Magnet 6 T Laser ----- IR multiphoton activation BIRD Blackbody IR Collisionally “In-beam” Nozzle-skimmer Capillary activation activated activation activation denaturation CAD dissociation CAD Also Tunable IR Laser for IR Photofragment Spectroscopy Electrospray Ionization of Bovine β-Casein 3 Variants, 5 Phosphorylations

Isotopic clusters A1 = 24007.2 -14 13C, 15N, 18O, 34S etc. 14 Da higher mass than A2 = 23968.2 -14 monoisotopic peak

Mass of highest Resolving Power 105 abundance peak A + Na (theoretical fit) 1 + 21H+ A + Na 2 B = 24077.2 -14 Electrospray Ionization

“Bottom Up”: trypsin digest, MS of peptides

Molec. Cellular Proteomics 2003, 2, 1253-1260. IDENTIFICATION

DNA predicted proteins: CAD Characterization of a Protein Mr = 16309.7

“Identification” by Bottom Up C

“Characterization” By Top Down Top-Down: 1) Select protein by MS/MS. 2) Don’t throw away connectivity info. Units: Amino Acids

X X

X X BUT completeLinear sequence Molecule, or exact Cleave PTM positioningOne Bond demands a fragment mass(neglecting from each interresidue ion chemistry) bond cleavage – and weakest bonds preferentially cleaved Chemistry of The Mass Spectrometry of Large Molecules

Electron ionization – requires vaporization Removes an electron, yields Odd-Electron Ion- More easily dissociated, less rearrangement

MS requires charged species- Chemical Ionization, Plasma Desorption, Fast Atom Bombardment, Matrix Assisted Laser Desorption Ionization, Electrospray Ionization But – these produce mainly Even-Electron Ions! New Chemical Reaction Needed M+ M+• How?

M+ + electron M+• Energetic Dissociation: CAD, IRMPD, BIRD, SID

Electron Capture Dissociation (with FTMS) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. JACS, 1998, 120, 3265 ~6 eV neutralization energy

N C

Electron Transfer Dissociation (Bottom-Up!) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J; Shabanowitz, J.; Hunt, D. F. PNAS, 2004, 101, 9528. Deamidation of Reduced Ribonuclease A

─NH →─OH, Exptl: 13,689.3-8 2 Calcd: 13,689.3-8 ∆ = +1 Da

Zabrouskov, Han, Welker, Zhai, Lin, van Wijk, Scheraga, McLafferty, Biochemistry 2006, 45, 987-992. MS/MS

Method to measure age? Deamidation of RNase A Only Asn67 found since 1968 Gln74

Asn34

Asn71,Asn94

Asn67 Electron Capture Nonergodic Dissociation <10-12 s, before energy randomization

• Negligible time for rearrangement • stops H/D scrambling (D atoms on exposed conformers) • Most interresidue bonds can be cleaved (250/258) e- neutralization energy >> bond dissociation energy • Fragment ion has N- or C-terminus, identifiable by CAD, ECD IRMPD Important for de novo sequencing

• Side chain posttranslational modifications are stable • Side chain posttranslational modifications are stable 26 Phophorylation Sites in β-Casein by Plasma-ECD

Phosphorylated126/208 Sites ininterresidue β-casein cleavages(24.9 kDa) by AI-ECD ya//yb /zc /

209 R E L E E L N V P G E I V E SSPPP L S SP E 20

189 E S I T R I N K K I E K F Q SP E E Q Q Q 40

169 T E D E L Q D K I H P F A Q T Q S L V Y 60 149 P F P G P I P N S L P Q N I P P L T Q T 80

129 PVVV PPFLQPEVMGVSKVKE 100

109 A M A P K H K E M P F P K Y P V E P F T 120

89 E S Q S L T L T D V E N L H L P L P L L 140

69 QSWMHQPHQPLPPTVMFPPQ 160

49 S V L S L S Q S K V L P V P Q K A V P Y 180

29 PQRDMPI QAFL LYQEPVLGP 200

9 V R G P F P I I V 209 b a c 129/208Sze, S. K.; bonds Ge, Y.; cleaved Oh, H. B.; total McLafferty, F. W. CAD ECD Anal. Chem. 2003, 75, 1599-1603. y z . y Electron Capture Nonergodic Dissociation <10-12 s, before energy randomization

• Side chain posttranslational modifications are stable • Tertiary noncovalent bonds not cleaved – ECD cleavage sites depend on conformation! Horn, Ge, McLafferty, Activated Ion Electron Capture Dissociation for Mass Spectral Sequencing of Larger (42 kDa) Proteins, Anal. Chem. 2000, 72, 4778-4784. Top Down Characterization of Carbonic Anhydrase "Top Down" of Bovine Carbonic Anhydrase 3.0 MW: calculated, 29024.7 -18, experimental, 29024.3 -18 ECD Mol. Wt.: calculated, 29024.7-18;-18, experimental, 29024.3-18-18 512 Fragment ion mass values, 183/258 interresidue bonds Fragment ions from residues 10-30 show 45 errors of -1 Da Fragment ions from residues 10-30 show 45 errors* * of -1 Da a• Protein Data Base: 10 31 b 2.5 Bovine (1) SHHWGYGKHDGPZHWHKDFPIANGERQSPVNIDTK c Sheep SHHWGYGEHNGPEHWHKDFPIADGERQSPVDIDTK y z• Human SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTH 2.0 ECD(2) SHHWGYGKHNGPZHWHKDFPIANGERQSPVDIDTK (1)Ref: Beu, Kelleher, S.; Senko, N.L.; Lin, M. H.Y.; W.; Valaskovic, Quinn, J. G.A.; P.; McLafferty,Aaserud, D.J.; F. Fridriksson, W. JASMS E.K.;1993, 4, 190 (2) Sze, McLafferty, S. K.; F.W.,Ge, Y.; J. Am.Oh, Chem. H. B.; Soc. McLafferty, 1999, 121, F. W.806-812. Anal. Chem. 2003, 75, 1599

O= 1.5 *

Product Ions Product Asp (D): -C-O- Σ -1 Da

%, %, vs. * O= Asn (N): H 1.0 -C-N- *** * *** 0.5 ** **

* * 0.0 20 40 60 80 100 120 140 160 180 200 220 240 Residue Automated MS Proteomics

Bottom-Up: 7000 proteins identified, M. Mann, et al., Nat. Methods, 2009, 6, 359.

Top-Down: Neil Kelleher, University of Illinois Off Line Separation: 1D Gel, RP LC, collect fractions MS: ESI, Octopole ion concentration, exact mass FTMS MS/MS: CAD, ECD Data Reduction: THRASH, ProSight PTM vs. predicted proteins Anal. Chem. 2004, 76, 197A-203A. Top Down in Chromatin Biology Nuclear membrane Chromatin fiber Interphase Chromatin fiber nucleus (30 nm dia.)

Prof. Neil H1 Nucleosomes Kelleher, U. of Illinois H1 Trans‐acting }factors Nuclear DNA pore

Nuclear matrix Top Down has provided the definitive list of histone isoforms present in yeast and human cells. What about a possible combinatorial “Histone Code”? Histone H3:

Ac Ac Me1 Me1 Ac Ac P Me1-3 Me1-3 P P Ac Ac P Ac Me1-3 P P Me1-3 ARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVKKPH…

TARKSTGGKacAPRKQL YRPGTVALR Kme2SphosTGGKacAPR TK QTAR me2 Kme3SAPSTGGVKme1KPHR KacQLATKacAAR KAARKme1SAPSTGGVKK

c P Me Me P A Ac P Ac Me P AR T K QTAR KS TGGKAPRKQLA TKAAR KSAP… 3 4 9 10 14 18 22 23 27 28

Can we use mass spectrometry to identify modifications on Single Forms that occur concurrently? High Resolution MS/MS on Histone H3 “Middle-Down” MS Proteomics

B.A. Garcia, J.J. Pesavento, C.A. Mizzen and N.L. Kelleher, Nature Methods, 2007, 4, 487.

M. Mann, N. L. Kelleher, PNAS, 2008, 105, 18132-18138.. Effect of Electrospray on Protein Conformation

Electrospray retains biological activity! Ions mass- separated in instrument, ACTIVE. Virus: Gary Siuzdak. Soft landing: Graham Cooks, Vicki Wysocki

Large (>0.5 MDa) Protein Complexes: Carol Robinson: Intraprotein noncovalent bonds stable.

Protein/Ligand Noncovalent Complexes: Steve Hofstadler, Nathan Yates, etc.: Noncovalent retention critical H/D Scrambling minimized by ECD: Cornell, Joe Loo

Top-Down of >50 kDa Proteins: “Activated ion” fails. Top Down Characterization of Carbonic Anhydrase "Top Down" of Bovine Carbonic Anhydrase 3.0 MW: calculated, 29024.7 -18, experimental, 29024.3 -18 ECD Mol. Wt.: calculated, 29024.7-18;-18, experimental, 29024.3-18-18 512 Fragment ion mass values, 183/258 interresidue bonds Fragment ions from residues 10-30 show 45 errors of -1 Da Fragment ions from residues 10-30 show 45 errors* * of -1 Da a• Protein Data Base: 10 31 b 2.5 Bovine (1) SHHWGYGKHDGPZHWHKDFPIANGERQSPVNIDTK c Sheep SHHWGYGEHNGPEHWHKDFPIADGERQSPVDIDTK y 25 ECD Spectra: 251/258 z• Human SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTH (2) 2.0 ECDinterresidue SHHWGYGKHNGPZHWHKDFPIANGERQSPV cleavagesDIDTK (1)Ref: Beu, Kelleher, S.; Senko, N.L.; Lin, M. H.Y.; W.; Valaskovic, Quinn, J. G.A.; P.; McLafferty,Aaserud, D.J.; F. Fridriksson, W. JASMS E.K.;1993, 4, 190 (2) Sze, McLafferty, S. K.; F.W.,Ge, Y.; J. Am.Oh, Chem. H. B.; Soc. McLafferty, 1999, 121, F. W.806-812. Anal. Chem. 2003, 75, 1599

O= 1.5 *

Product Ions Product Asp (D): -C-O- Σ -1 Da

%, %, vs. * O= Asn (N): H 1.0 -C-N- *** * *** 0.5 ** **

* * 0.0 20 40 60 80 100 120 140 160 180 200 220 240 Residue hydrophobic bonding Breuker, McLafferty PNAS, 2008, 105, 18151 >75 kDa proteins, negliglble topdown dissociation

hydrophobic bonding “Prefolding Dissociation” Prefolding Dissociation

Protein molecular ion intractability above 50 kDa

Han, Jin, Breuker, McLafferty Science, 2006, 314, 109-112 Top Down PFD of a 200 kDa Protein

All 27 Cysteines assigned as S-H or S-S Human Complement C4

87 fragment ion masses Specify 4 previously unidentified intrachain S-S bonds (no glycosylation) 57% overall sequence Localize 6 Cys residues of three interchain S-S bonds coverage Denaturing Refolded Proteins in the Ion Cell

IR photoexcitation Breuker, Oh, Horn, Cerda, McLafferty, J Am Chem Soc 2002, 124, 6407–6420.

Ubiquitin 7+ Ions

IR laser pulse + (Ser8 + 8H) : Prebiotic Chiral Selection? S.C. Nanita, R.G. Cooks, Angew. Chem. Int. Ed. 2006, 45, 554-569.

+ • D,L-Ser selectively yields (D-Ser8 + H) and (L-Ser8 + H)+.

• serine octamers are also selectively formed as neutrals and anions

• serine octamers are also selectively formed from solid and solution phases.

Electrospray retains the conformer structure? Infrared Photodissociation Spectroscopy Oh, Breuker, Sze, Ge, Carpenter, McLafferty Proc. Natl. Acad. Sci. USA 2002, 99, 15863 Oh, Lin, Hwang, Zhai, Breuker, Zabrouskov, Carpenter, McLafferty, JACS, 2005, 127, 4076

OPO laser power : 6-10 mJ/pulse H 4076-4083 — 127, 2005, -CHRO H JACS, — 2.7 µm

=O -CO H —

-NH X X •••• 3.0 •••• H brouskov, Carpenter, McLafferty, brouskov, H — — N -O IRPDS Peak Assignments Serine Dimer Oh, Lin, Hwang, Zhai, Breuker, Za Zhai, Breuker, Oh, Lin, Hwang, -1 AbsorptionSerine atOctamer 3675 cm : None: Hwang, Lin, Oh, Breuker, S.C. Nanita, R.G. Cooks, Angew. Chem. Carpenter, McLafferty, ASMS,2004. Int. Ed. 2006, 45, 554-569. Strong: Kong, Tsai, Sabu, Han, Y.T. Lee, Chang, Tu, Kung, Wu, Angew. Chem. 2006, 45, 4130. Mazurek, Geller, Lifshitz, McFarland, H/D exchange Marshall, Reuben, J. Phys. Chem. A 2005, 109, 2107.

A – isomer -- B SWIFT, then Isomer B H/D exchange chirally inactive

CAD, then H/D exchange

SWIFT, CAD, H/D eschange Dissociation: (1) iv → products; (2) v → products. Isomerization: (3) iv → i; (4) v → vi. Dissociation: (5) i → products (6) vi → products.

IRPD: at least 6 isomers SWIFT

CAD 0 kJ/mol Free O-H

CAD of II yields I -13.5 kJ/mol

Xianglei Kong, Cheng Lin, -11.9 kJ/mol Giuseppe Infusini, Han- Bin Oh, Honghai Jiang, Kathrin Breuker, Chih-Che Wu, Oleg P. Charkin, Huan-Cheng Chang, and Fred W. McLafferty* , ChemPhysChem, 2009. IRPD Spectra of Protein Ions

CF3SO2OH adduct of Mellitin, 2.7 kDa

Loss of O-H, N-H stretch region

CF3SO2OH

No O-H, no side-chain N-H or N+-H; only amide N-H O-H, N-H red-shift by H-bonding S/N ~200!

Similar spectra from 8.6, 12.3, and 13.7 kDa proteins ------AND MOST CHARGE STATES Breuker, K.; Oh, H. B.; Lin, C.; Carpenter, B. K.; McLafferty, F. W. Proc. Natl. Acad. Sci. USA 2004, 101, 14011-14016. Tertiary Structures of Gaseous Ubiquitin Ions K. Breuker, H.-B. Oh, D. M. Horn, B. A. Cerda, F. W. McLafferty JACS 2002, 124, 6407-6020.

Crystal Structure

H+

+ Hydrophobic IRH Photofragment Spectra

7+ 7+ bonds7+ unfold 30 (U + 7H) /(U + 7H+ TA) Solution25 20

/(U + 7H + TA) 15

unfolding7+ Hydrogen 10 5 bonds stronger (U + 7H) 0

3100 3200 3300 3400 -1 wavenumber (cm ) Gaseous conformers, per ECD

Acknowledgments Kent Henry, Evan Williams, Bing Wang, Jorge Furlong, John Quinn, Joe Loo, Mike Senko, Steve Beu, Russ Chorush, Dan Little, Neil Kelleher, Paul Speir, Troy Wood, Peter O’Connor, Z. Guan, Gary Valaskovic, Dave Aaserud, Roman Zubarev, Dave Horn, Einar Fridriksson, Nate Kruger, Mark Lewis, Petia Shipkova, Blas Cerda, Ying Ge, Kathrin Breuker, HanBin Oh, Newman Sze, Huili Zhai, Mariam ElNaggar, Vlad Zabrouskov, Harold Hwang, Cheng Lin, Xuemei Han, Guiseppe Infusini, Honghai Jiang, Xianglei Kong, Mahmud Hossain, Sergio Castro Tadhg Begley, Barbara Baird, Barry Carpenter, Harold Scheraga, Klaas van Wijk, Floyd Davis General Medical Institute, National Institutes of Health