Joshua F. Emory and Scott A. McLuckey

1. Transmission Mode Ion/Ion Reactions in the Q0 cell of a Hybrid Triple Quadrupole/Linear Ion Trap Instrument Joshua F. Emory and Scott A. McLuckey Department of Chemistry, Purdue University, West Lafayette, IN 47907

2. Overview • The possibility of performing transmission mode ion/ion reactions in the higher pressure RF only Q0 quadrupole is being investigated. • The ability to perform these proton transfer charge reduction or charge inversion reactions in Q0 would enable multiple ion/ion reactions to be performed in separate regions of the instrument, perhaps by doing a preparatory reaction in Q0 and a second reaction in Q2. • The drawback to performing ion/ion reactions in the Q0 cell is that neither the analyte nor the reagent ion can be isolated prior to introduction into the Q0 quadrupole and the subsequent ion/ion reaction.

3. Introduction • Proton transfer ion/ion reactions have been used for protein charge reduction, to concentrate the charge envelope of proteins as well as to invert the polarity of peptides within the mass spectrometer. • These reactions can be performed by storing the reagent in the Q0 cell and passing the analyte through the Q0 cell or by storing the analyte in the Q0 cell and passing the reagent through the Q0 cell.

4. Experimental • Perfluoro-1-octanol (PFO), the charge reduction reagent, solution consisted of a aqueous solution of ~200 to 400 µM of reagent in (48.5:48.5:3) methanol, water, and ammonium hydroxide. • Protein electrospray solutions were diluted to ~10 to 20 µM in (49:49:2) water, methanol, and acetic acid. • The charge inversion reagents, polypropylenimine diamino-butane (DAB) dendrimers and polyamidoamine (PAMAM) dendrimers were made in aqueous ~1-2% acetic acid and in aqueous ~1-2% ammonium hydroxide, respectively. • Bradykinin electrospray solutions consisted of an aqueous solution of ~10 µM concentration of peptide in (49:49:2) methanol, water, and ammonium hydroxide.

5. Modified Q-Trap 2000/4000 Transmission mode reactions in Q0 can be performed by: By storing the reagent ion in Q0, and transferring the desired analyte ion through the Q0 cell. By storing the desired analyte ion in Q0, and transferring the reagent ion through the Q0 cell. Q0 Q1 Q2 Q3 Auxiliary RF applied for ion parking Dual ESI sources N2 CAD Gas Aux AC RF Exit lens Orifice Skimmer IQ1 ST IQ2 IQ3 Deflector Triggered +HV Triggered -HV Ion/Ion Reaction Detector

6. Mutual Storage Mode Ion/Ion Reactions in a LIT - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - - - - - - + + + + + + + + + + end lens end lens LIT Trapping Anions Trapping Cations RF • Mutual storage of oppositely charged ions requires application of auxiliary RF to the end lenses of the quadrupole. Transmission Mode Ion/Ion Reactions in a LIT Passing Anions TrappingCations DC Passing Cations Trapping Anions DC • Does not require application of auxiliary RF to end lenses. • Performing ion/ion reactions in Q0 allows further ion/ion reactions or CID to be performed in the Q2 or Q3 cell of the instrument.

7. Transmission Mode Charge Inversion • Involves transfer of multiple protons to an analyte or the removal of multiple protons from an analyte. • Charge inversion of an analyte species separates the ionization polarity of the analyte from analysis polarity. • Separation of ionization polarity from analysis polarity allows interrogation of an analyte in both polarities, increasing the amount of information that can be obtained from the analyte via dissociation methods such as CID and ETD.

8. m+ n- + - - 2+ + H - H - H + 2H Charge Inversion Process: Multiple Proton Transfer Polyamidoamine (PAMAM) Dendrimer Peptide Ion (Bradykinin) 1,4-Diaminobutane (DAB) Dendrimer

9. Charge Inversion of Bradykinin -1 in Q0 100 100 Bradykinin -1 Gen 4 DAB +6 + 50 50 900 1100 300 700 500 700 800 900 1000 1100 1e+7 Passing bradykinin -1 through Q0 for 200 ms Bradykinin +2 Bradykinin +1 5e+6 DAB +6 DAB +5 DAB +3 DAB +4 700 1100 1300 1500 300 500 900 m/z

10. Charge Inversion of Bradykinin +1 in Q0 100 100 + PAMAM 50 50 Bradykinin +2 Bradykinin +1 500 2000 300 500 700 900 1100 0 1000 1500 8e+6 Passing bradykinin +2/+1 through Q0 for 1 s Bradykinin -1 PAMAM -4 PAMAM -5 4e+6 PAMAM -6 Bradykinin -1 1200 800 1000 400 600 m/z

11. Quadrupole Rod A. Ion Parking in Q0 • Application of a Dipolar AC frequency to one pair of rods in a quadrupole • Analogous to low amplitude CID • Parking has previously been performed in 3D ion traps and in the Q2 cell of 2D traps.

12. Types of Ion Parking Quadrupole Rod A) SFOR ion parking where a single-frequency, on resonance, low amplitude ac signal is applied to an opposing quadrupole rod set. B) HALF parallel ion parking where a high-amplitude, low-frequency ac signal is applied to an opposing quadrupole rod set. A. f0,z (21/2/ro2 Ω)(zV/m) = c(zV/m) B. ωz Ω

13. Ion/Ion Reaction of Insulin with PFO in Q0 Insulin +2 Insulin +3 Insulin +4 100 100 +5 Insulin -1 PFO perfluoro-1-octanol + +6 50 50 CF3(CF2)6CH2OH +4 500 700 900 1100 1300 1500 1700 300 400 500 600 200 2e6 No Ion Parking 1e6 4e7 Insulin +4 SFOR Ion Parking of Insulin +4 f = 32.633 kHz, 4 Vp-p 2e7 Insulin +3 3000 1000 1400 1800 2200 2600 3400 m/z

14. 1000 ESI of Protein Mixture: Insulin, Bovine Cyt. C Equine Cyt. C, Ubiquitin Myoglobin, Lysozyme x3 500 600 1000 1400 Parallel Ion Parking of a Protein Mixture in Q0 150 U+2 100 50 I+2 * * * 600 U+3 Insulin = I Ubiquitin = U Equine Cyt. C = E Bovine Cyt. C = B Myoglobin = M Lysozyme = L B+4 I+2 400 M+6 L+5 M+5 E+4 200 E+3 L+4 B+5 E+5 B+3 I+3 U+2 U+4 0 2000 2400 2800 3600 4400 3200 4000 m/z

15. Results • Charge reduction by proton transfer with or without ion parking of the desired charge state can be performed in the Q0 cell. • Charge inversion of bradykinin -1 to +2/+1 and charge inversion of bradykinin +2/+1 to bradykinin -1 can also be performed in the Q0 RF only quadrupole. • The use of Q0 as a reaction cell enables two reactions to be performed in rapid succession in separate regions of the instrument. For example, two ion/ion reactions could be performed on an analyte using transmission mode experiments, eliminating the need for auxiliary RF on the end lenses of the Q0 or the Q2 quadrupoles.

16. Conclusions • Transmission mode proton transfer charge reduction reactions can be performed in the Q0 quadrupole, eliminating the need for application of auxiliary RF on the end lense of the Q0 quadrupole. • Charge inversion of bradykinin from negative to positive and from positive to negative in the RF only Q0 cell has been demonstrated. • Separates analysis polarity from ionization polarity • Performing ion/ion reactions in Q0 allow further ion/ion reactions to be performed in the Q2 and/or Q3 cell of a hybrid linear ion trap. • HALF and SFOR methods of ion parking have been successfully demonstrated in the RF only Q0 cell.

17. References • M. He, J.F. Emory, S.A. McLuckey, Anal. Chem., 77 (2005) 3173-3182. • M. He, S.A. McLuckey, J. Am. Chem. Soc., 125 (2003) 7756-7757. • P. A. Chrisman, S. J. Pitteri, S. A. McLuckey, Anal. Chem., 78 (2006) 310-316. • P.A. Chrisman, S.J. Pitteri, S.A. McLuckey, Anal. Chem., 77 (2005) 3411-3414.

18. MDS SCIEX NIH GM 43572 McLuckey Group Acknowledgments