Top-Down Sequencing and Charge-Reduction Mass Spectrometry of Proteins in their Native State

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1 Top-Down Sequencing and Charge-Reduction Mass Spectrometry of Proteins in their Native State Jonathan P. Williams, Ph.D Waters Corporation, Manchester, UK Health Sciences Research 214 Waters Corporation 1

2 Outline The utility of ETD and charge reduction for top down native MS Introduction to the SYNAPT G2-Si including ETD for top down native MS Overview of top down native MS Limitations of native MS (CID) for characterisation of protein complexes (Hemoglobin example) Advantages of native MS (ETD) for characterisation of protein complexes (Alcohol Dehydrogenase (ADH) example) Native MS & charge reduction with protein standards Further example of native MS ETD & charge reduction Summary 214 Waters Corporation 2

3 Native MS analysis of the Chaperonin Protein Complex GroEL from E. coli using the Synapt G2-S: 82 kda % GroeL Purge gas 5 cone gas 5 JPW_KG_3 25 (.44) Cm (2:28) 1 Intact 14-mer Exquisite Speed & Sensitivity 2 heptameric rings of ~57 kda subunits stacked back-to-back TOF MS ES+ 1.94e X-ray crystallography models from two different views of GroEL (taken from Waters Corporation

4 Native MS/MS analysis of the Chaperonin Protein Complex GroEL from E. coli using the Synapt G2-S % JPW_KG_2 1 (.34) Sm (SG, 2x1.); Cm (1:28) monomer TOF MS ES+ 1.12e mer Intact 14-mer mer Waters Corporation 4

5 Nomenclature for peptide sequencing by Tandem Mass Spectrometry There are 6 possible fragment ions for each amino acid residue The a, b, and c ions having the charge retained on the N-terminal fragment, and the x, y, and z ions having the charge retained on the C-terminal fragment The most common cleavage sites are at the CO-NH bonds which give rise to the b/y ions or the N-Cα bonds which give rise to the c/z ions CID ETD 214 Waters Corporation 5

6 Earlier investigations JMS,1998, 33, JASMS,21, 21, Waters Corporation 6

7 SYNAPT G2-Si - ETD enabled 214 Waters Corporation 7

8 Chamber Containing ETD Reagent Reagent chamber 214 Waters Corporation 8

9 T-Wave effects on ETD and charge-reduction reaction dynamics.2-.4v Lower T-Wave amplitudes ETD Volts.7-1.5V Higher T-Wave amplitudes ETD C-R Increase ion energy for charge-stripping either from exit of Trap or upon entry into Transfer Volts 214 Waters Corporation 9

10 Native mass spectrometry -ETD of ADH tetramer (25+) P Frank Sobott et. al. J. Am. Soc. Mass Spectrom. 25 (214) A. Native MS of ADH B. ETD product spectrum of 25+ charge state Charge reduced species ETD fragments C. ETD product spectrum of low range D. Sequence of a ADH monomer with sites of cleavage highlighted in red E. X ray crystallography structure of ADH 214 Waters Corporation 1

11 Native mass spectrometry & charge reduction - Examples using protein standards Ubiquitin Cytochrome C Myoglobin 214 Waters Corporation 11

12 % Native mass spectrometry - MS analysis of non-covalent protein complexes - Charge distribution of hemoglobin (Hb) tetramer Name Mutation Symtoms (homozygote or heterozygote) 1 A Hb S β6 (Glu to Val) SCD or asymtomatic Hb C β6 (Glu to Lys) mild anemia or asymptomatic Hb E β26 (Glu to Lys) thalassemia minor or mild microcytosis Hb (normal adult~ 97%) is a tetrameric structure consisting of: Hb D-Punjab β121 (Glu to Gln) mild anemia or asymptomatic Hb O-Arab β121 (Glu to Lys) mild anemia or asymptomatic - 2 x -chains (141 A.A s) each of mass 15,126 Da - 2 x -chains (146 A.A s) each of mass 15,867 Da - 4 x heme groups each of mass Da A ~ 1 A A Waters Corporation 12

13 % Native mass spectrometry - Collision induced dissociation (CID) of Hb tetramer Hb TETRAMER TMS Precursor ion 379, CE 42eV TRAP Trap CE 42eV Mixed monomers Precursor ion heme Mixed trimers Waters Corporation 13

14 % Native mass spectrometry -Electron transfer dissociation (ETD) of Hb tetramer ([M+18H] 18+, 3581) Hb TETRAMER ETD M/Z Hb α-chain sequence information following ETD c'' c'' c'' c'' c'' c'' c'' Waters Corporation

15 % Native mass spectrometry -Electron transfer dissociation (ETD) of Hb tetramer ([M+18H] 18+, 3581) Hb TETRAMER ETD M/Z Hb β-chain sequence information following ETD This method can be used for phenotyping. c'' Whole blood sample analysed as received for clinically significant variants ~ 1-15mins c'' c'' c''2 c'' c'' c'' c'' Waters Corporation 15

16 % Native mass spectrometry - Electron transfer dissociation (ETD) of Hb tetramer ([M+17H] 17+, 3795) Precursor ion ETD backbone product ions Transfer CE 3eV c''119 2+; Waters Corporation 16

17 % Native mass spectrometry - Electron transfer dissociation (ETD) of Hb tetramer ([M+17H] 17+, 3795) : charge reduction ETD backbone product ions Transfer CE 3eV and Bias 5V Precursor ion Waters Corporation 17

18 % Native mass spectrometry - Electron transfer dissociation (ETD) of Hb tetramer ([M+17H] 17+, 3795) : further charge reduction ETD backbone product ions Transfer CE 3eV and Bias 15V Precursor ion Waters Corporation 18

19 % % ZOOM mass spectrum of ETD and charge-reduced ADH ADH OBS ms adh in ETD mode MS A26 A A Waters Corporation 19

20 % % ZOOM mass spectrum of charge-reduced Tetramer of Concanavalin A (Jack Bean) ETD 515 WH.25V, CE 14V, 3mL/min, bias 11V, MS bias 1mg/mL 11V Ammonium Acetate Concanavalin A (Sigma L7647) MS A Waters Corporation 2 1+

21 % % ZOOM mass spectrum of charge-reduced Pyruvate Kinase MS A Waters Corporation 21

22 Summary Fragmentation of Native protein CID provides sub-unit information ETD provides sequence information for further structural elucidation of protein complexes Electron transfer reaction produces singly charged ions useful for exact mass measurements and protein micro-heterogeneity characterisation SYNAPT G2-Si TOF mass analyser shown to have a high ion transmission which is important for charge reduction mass spectrometry Ion mobility can also provide information about protein complex conformations Future work Analysis of heterogeneous glycoproteins such as recombinant mabs that possess a number of different glycoforms and PEGylated bioconjugates Measure of the CCS of the charged reduced species 214 Waters Corporation 22

23 Acknowledgments - Jeff Brown-Waters - Health Sciences Group-Waters - Professor Frank Sobott and Frederik Lermyte (Antwerp University) 214 Waters Corporation 23

24 ETD Overview ETD is an alternative and complementary dissociation technique to CID. Peptides are dissociated by electron transfer from a donor chemical anion, to positively charged cations (peptides, proteins), leading to an inherently similar fragmentation process to ECD. The nature of the fragmentation process generally leads to a balanced spectrum with similar abundances for many fragment ions. ETD is beneficial for the analysis of molecules with labile bonds which are preferentially cleaved by CID. 214 Waters Corporation 24

25 Intact Monoclonal Antibody: Top-Down ETD Waters Intact mab Standard 214 Waters Corporation 25

26 Intact mab Waters Standard ETD of [M+57H] 57+ Precursor ion [M+57H] 57+ mixture of c- and z-type ions from LC and HC charge-reduced species 214 Waters Corporation 26

27 Intact mab Waters Standard ETD of [M+57H] 57+ Zoom at a particular range Data very complex. Overlapping charge states 214 Waters Corporation 27