Overview

1. Overview • 2,6-naphthalene dicarboxylic acid (NDCA), ethylene diamine tetracetic acid (EDTA), and perfluoro-1,10-decane diol (PFDO) were used to charge invert CID product ions from several peptides from +1 to -1. • Hypothetically, y-ions should charge invert from +1 to -1 more readily than b-ions, because y-ions have a carboxylate group on the C-terminus and b-ions do not. • Charge inversion likely occurs by the formation of a long-lived chemical complex2. • Selective charge inversion should depend on the differences in proton affinity of the b-ion and y-ion.

2. Introduction • Using ion/ion reactions for charge inversion eliminates the need for high collision energies and gives product ions with little or no fragmentation1. • Differentiating y-ions from b-ions in CID spectra is useful for peptide and protein identification. • Identifying y-ions and b-ions in a CID spectrum without prior knowledge of the peptides’ sequence is tedious. • Charge inversion via ion/ion reactions is a desirable way todifferentiate between y-ions and b-ions because of the experimental control available. • Distinguishing y-ions from b-ions by charge inversion would facilitate sequencing and identification of unknown proteins.

3. Approach • CID product ions of one of several peptides were charge inverted from +1 to -1 using three doubly charged negative reagent ions. • The relative proton affinities of y-ions and b-ions should play an important role in charge inversion. • The charge state dependent proton affinity of the reagent ion is an important factor in selective charge inversion of y-ions versus b-ions. • Selectively charge inverting y-ions from +1 to -1would make differentiating between y-ions and b-ions in CID spectra of unknown proteins a real possibility.

4. Peptides PPGFSPFR RPPFSPFR RPPGFSPFR VDPVNFK ALILTLVS YGGFL Reagent Ions Experimental • Mass spectrometer: modified QTRAP 20003 • Ionization source: pulsed dual nano-ESI source EDTA NDCA PFDO

5. CID ExperimentalSequence 2. Peptide CID 1. Isolate peptide +1/+2 +1 CID Product Ions 4. Charge Inverted CID 3. Isolate Reagent 4. Ion/Ion Charge Inversion Reaction -1 -2

6. IQ2 IQ3 EX triggered -/+ HV Q1 Q2 Q3 Nano-ESI tip Nano-ESI tip triggered +/- HV ~ ~ ~ Modified QTRAP 2000 Q0 Pulsed Dual Nano-ESI Source

7. Negative Reagent Ions PFDO EDTA NDCA

8. B-ion/Y-ionCalculations • Density Functional Theory calculations [UB3LYP 6-31+G(d)] were performed on a model y1 ion and b2 ion4. • The proton affinities of the model y-ion and b-ion were calculated as shown below. PA = -ΔHrxn = -[ΔHf (Anion) + ΔHf (H+) – ΔHf (Neutral)]

9. B-ion/Y-ion Structures B2 Anion Y1 Anion B2 Neutral Y1 Neutral

10. Theoretical Predictions • When the reagent ion’s proton affinity is between the proton affinity of the y-ions and b-ions, y-ions were predicted to charge invert more efficiently: • Only y-ions have a C-terminal carboxylate group allowing y-ions to charge invert more easily from +1 to -1. • Calculations show the proton affinity of the b2 ion from diglycine is 6 kcal/mol greater than for the y1 ion of glycine. • Charge inversion selectivity is expected to be dependent on the acidity of the reagent ion.

11. Experimental Results • All three positive to negative charge inversion reagents charge inverted CID product ions from all the peptides tested. • PFDO charge inverted CID product ions by complex formation only. • Both EDTA and NDCA charge inverted CID product ions by proton transfer only. • Both y-ions and b-ions were charge inverted by all three reagents, and none of the reagents selectively charge inverted y-ions versus b-ions.

12. 1.3e7 6.5e6 700 800 900 1000 1100 1200 500 600 1.3e7 x2 6.5e6 1300 350 450 550 650 750 850 950 150 250 PPGFSPFR CID and charge inversion with PFDO M+2 y1 *= -NH3 or –H2O CID spectrum of Des R1 bradykinin PPGFSPFR y1* Positive Mode y3*, y3 b4 y4* y4 y6* y5 y6 y7 y1*+P Charge Inversion with PFDO by complex formation x2 P = PFDO - y1 + P - Negative Mode y3* + P y3 + P *= -NH3 or –H2O y4* + P y6* + P y4 + P y6 + P y7 + P y2 + P y2* + P y5 + P m/z

13. 4e7 2e6 350 750 850 950 550 650 450 y6 M b8, M* 2e6 b7 b5, y4*, y4 y6* b7+H2O b8* M+Na y5 y3 y7 350 750 850 950 550 650 450 PPGFSPFR CID and charge inversion with NDCA y6 CID spectrum of Des R1 bradykinin PPGFSPFR *= -NH3 or –H2O Positive Mode b8, M* b7+H2O M b7 b5, y4*, y4 y6* b8* y5 y3 y7 M+Na 4e7 *= -NH3 or –H2O Charge inversion using 2,6-NDCA Negative Mode m/z

14. 4e7 2e6 350 750 850 950 550 650 450 m/z PPGFSPFR CID and charge inversion with EDTA y6 CID spectrum of Des R1 bradykinin PPGFSPFR *= -NH3 or –H2O Positive Mode b8, M* b7+H2O M b7 b5, y4*, y4 y6* b8* y5 y3 y7 M+Na M Charge inversion using EDTA y6 *= -NH3 or –H2O x2 Negative Mode b7+H2O b8, M* b8* b7 y6* b5, y4*, y4 y5 y3 y7 M+Na

15. Rel. Ab. Post Chg Inv. Charge Inversion Ratio = Rel. Ab. Pre Chg Inv. Charge Inversion of Des R1 (PPGFSPFR) with NDCA, EDTA, and PFDO Charge Inversion Ratio

16. Rel. Ab. Post Chg Inv. Charge Inversion Ratio = Rel. Ab. Pre Chg Inv. Charge Inversion of VDPVNFK CID with NDCA, EDTA, and PFDO x2 Charge Inversion Ratio

17. Rel. Ab. Post Chg Inv. Charge Inversion Ratio = Rel. Ab. Pre Chg Inv. Charge Inversion of ALILTLVS CID with NDCA, EDTA, and PFDO x10 x4 Charge Inversion Ratio

18. Discussion • Overall y-ions were not charge inverted more selectively than b-ions using NDCA, EDTA, and PFDO. • NDCA has a higher overall charge inversion efficiency than EDTA or PFDO for all ions. • NDCA does not appear to preferentially charge invert any particular type of ion. • PFDO may have some preference for ions containing basic residues [Lys (K) and Arg (R)] and acidic residues [Glu (E) and Asp (D)]. • EDTA discriminates against the sodiated parent ion in the charge inversion process.

19. Discussion • NDCA charge inverts b-ions and y-ions although NDCA’s calculated 2nd proton affinity was lower than the proton affinities of the model b-ion and y-ion (suggesting that NDCA would not charge invert b or y-ions). • The b-ions and y-ions studied experimentally likely had lower proton affinities of NDCA, EDTA, and PFDO because the experimentally studied b-ions and y-ions were composed of amino acids with polar groups that lowered the proton affinity of the b-ions and y-ions. • Functional groups and amino acid composition will affect the relative proton affinities of b-ions and y-ions.

20. Conclusions • PFDO, NDCA, and EDTA are can be used as positive to negative charge inversion reagents by complex formation and by proton transfer. • Energy lowering interactions in the b-ions and y-ions studied experimentally probably prevented NDCA, EDTA, and PFDO from selectively charge inverting y-ions versus b-ions. • Factors such as the presence of acidic or basic residues also may play a role in charge inversion selectivity. • Selective charge inversion of y-ions rather than b-ions using these reagents is not possible, and other reagents or approaches are being investigated.

21. Acknowledgments • Research sponsored by the Office of Basic Energy Sciences, Division of Chemical Sciences under Award No. DE-FG02-00ER15105. • Amy Facility at Purdue University.

22. References • 1. M. He, J.F. Emory, S.A. McLuckey, Anal. Chem., 77 (2005) 3173-3182. • 2. J.M. Wells, P.A. Chrisman, S.A. McLuckey, J. Am. Chem. Soc., 125 (2003) 7238-7249. • 3. Y. Xia, X. Liang, S.A. McLuckey, J.Am. Soc. Mass Spectrom.,16 (2005) 1750-1756. • 4. B. Paizs, S. Suhai, Mass. Spectrom. Rev., 24 (2005) 508-548.