5 Data Analysis
The data was manually analysed using the Xcalibur software. Backbone c, y and z ions were assigned. Analysis included any peaks showing a loss or addition of water (+/- 18), phosphate (+/- 80) and phosphoric acid (+/- 98). Neutral loss peaks were determined genuine if their mass accuracy was between 0 and 10 ppm. For those peptides in which losses of 18, 80 or 98 Da were observed, the intensity of the peaks (y axis) was plotted against supplemental activation (x axis) to determine if there is a relationship between the losses and the activation energy. This allowed us to set the parameters for the FAIMS analysis so that if any phosphate relocation were observed, it could not be a consequence of the activation energy.
The FAIMS data were also analysed manually: Each spectrum was labelled and the peaks were assigned, again noting any peaks corresponding to neutral losses of -18, -80 or -98 Da.
Results and Discussion
All 9 peptides were subject to ETD at varying supplemental activation energies in the mass spectrometer. The peaks in each spectrum were assigned and any neutral loss peaks plotted against activation energy. Of the peptides studied, only peptide 9 showed any ambiguous fragments (c8-80). The c8-80 fragment of peptide 9 has identical mass to the c8 fragment from peptide 8. Figure 1 shows a of plot of peak intensity against activation energy for the c8 and c8- 80 fragments from peptide 9. There is no loss of 80 Da until a supplemental activation energy of 12.5%, at which point the peak intensity rapidly increases up to an activation energy of 25%.
Figure 1 - APLsFLGSLPKSYVK,3+,c8-80
20000 40000 60000 80000 100000 120000 140000 160000
0 0 5 10 15 Supplemental Activation
Figure 1. Effect of supplemental activation on the abundance of the c8 and c8-80 fragments of triply-charged peptide 9.
Figure 2 shows the saETD mass spectra obtained for peptide 9 at supplemental activations of 0%, 15% and 25% respectively. The c8-80 fragment is not present at activation energy 0%, however it is apparent when this is increased to 15% and its abundance further increases at 25%. Again you can see from the fragmentation patterns that the number of fragments increases with increasing activation energy. This shows that when considering optimum activation energy, we should not make it too low as we will not see efficient fragmentation.
From the LTQ Orbitrap Velos ETD results, we determined that when carrying out the FAIMS experiment we would use a maximum supplemental activation of 7.5%, as at this activation energy for all of the peptides we do not see any loss peaks. This then allowed us to look at the FAIMS data and analyse any neutral loss peaks knowing that they were solely due to the FAIMS and not saETD. The dispersion voltage was varied because it may cause low levels of collisional activation through collisions with the carrier gas. By reducing the DV, the ions path are slowed reducing the energy transferred.
The FAIMS ETD spectra were analysed for the presence of ambiguous fragments, e.g., neutral loss peaks. The previously identified c8-80 fragment from peptide 9 was not present in the FAIMS data at any of the 3 dispersion voltages, e.g, see Figure 3. No ambiguous fragments were observed in any of the FAIMS data. This result informs us that the FAIMS device does not promote the loss of the phosphate and that in this is example it is solely down to the supplemental activation. This knowledge means that we can confidently carry out experiments using the FAIMS and accurately identify the phosphorylated residues of co-eluting isobaric phospho-peptides.
(a) 100
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
5 0 c3 200 400 600 m/z 800 1000 1200 x5 [M+3H]3+ c ions
A P L S F L G S L P K S Y V K L
L L L
[M+2H]2+ c12 L L L
L L L L
[M+H]+
z ions y ions
x5 (b) x5 100 c8 c5 c7 z8• z10• [z13•]2+ z3• c4 z4• y5 c6 y12 z9• z12• 1400 1600 c11 z11 c13 z13 c14
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
0 5
200 c3 400 [M+3H]3+ x5 20 25 30 References
[1] Edelson-Averbukh, M., et al., Gas-Phase Intramolecular Phosphate Shift in Phosphotyrosine-Containing Peptide Monoanions. Analytical Chemistry, 2009. 81(11): p. 4369-4381.
[2] Viglietto, G., et al., Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer. Nature Medicine, 2002. 8(10): p. 1136-1144.
[3] Syka, J.E.P., et al., Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America, 2004. 101(26): p. 9528-9533.
[4] Zubarev, R.A., N.L. Kelleher, and F.W. McLafferty, Electron capture dissociation of multiply charged protein cations. A nonergodic process. Journal of the American Chemical Society, 1998. 120(13): p. 3265-3266.
[5] Sweet, S.M.M., et al., Large Scale Localization of Protein Phosphorylation by Use of Electron Capture Dissociation Mass Spectrometry. Molecular & Cellular Proteomics, 2009. 8(5): p. 904-912.
[6] Creese, A.J. and H.J. Cooper, The effect of phosphorylation on the electron capture dissociation of peptide ions. Journal of the American Society for Mass Spectrometry, 2008. 19(9): p. 1263-1274.
[7] Coon, J.J., et al., Electron transfer dissociation of peptide anions. Journal of the American Society for Mass Spectrometry, 2005. 16(6): p. 880-882.
[8] Coon, J.J., et al., Activated-Ion Electron Transfer Dissociation Improves the Ability of Electron Transfer Dissociation to Identify Peptides in a Complex Mixture. Analytical Chemistry, 2010. 82(24): p. 10068-10074.
[9] Coon, J.J., et al., Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors. Analytical Chemistry, 2007. 79(2): p. 477-485.
[10] Xuan, Y., et al., High-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled with high- resolution electron transfer dissociation mass spectrometry for the analysis of isobaric phosphopeptides. Rapid Communications in Mass Spectrometry, 2009. 23(13): p. 1963-1969.
[11] Guevremont, R., High-field asymmetric waveform ion mobility spectrometry: A new tool for mass spectrometry. Journal of Chromatography A, 2004. 1058(1-2): p. 3-19.
[12] Palumbo, A.M. and G.E. Reid, Evaluation of Gas-Phase Rearrangement and Competing Fragmentation Reactions on Protein Phosphorylation Site Assignment Using Collision Induced Dissociation-MS/MS and MS3. Analytical Chemistry, 2008. 80(24): p. 9735-9747.
(c) 100 c12 [M+2H]2+ c5 [M+2H]2+-49 c7 z8• c11 z11 [z13•]2+ z3• c4 c6 z4• y5 600 800
c8-80 z7•
z9• y9 1000 m/z 1200 y12 z12• 1400 1600 c13 z13 c14 z10• [M+H]+ c8
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
0 5
c3 200 400 600 m/z Figure 2. saETD mass spectra of triply charged peptide 9 (APLpSFLGSLPKSYVK), supplemental activation of (a) 0%, (b) 15%, and (c) 25%. 800 [M+3H]3+ c ions
A P L S F L G S L P K S Y V K L
LL L
L L c5
L L
L L c12
L L
L
z ions y ions
x5 100
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
0 5
200 c3 400 600 [M+2H]2+ [M+3H]3+
z3• c4 z4• c5 [z13•]2+ y5 c6 800 m/z c7 z7• c8 z8• z9• y9 1000 1200 z10• c11 z11 z12• y12 1400 c13 z13 c14 1600 c12 [M+H]+
Figure 3. FAIMS ETD mass spectrum triply charged peptide 9 (APLpSFLGSLPKSYVK) at dispersion voltage of 5000.
Conclusion
From this work it can be concluded that the optimum supplemental activation to carry out ETD is at 7.5, to give enough energy so that there is significant fragmentation, but to inhibit the loss or transfer of the phosphate group. Our results suggest that if the FAIMS ETD is carried out at with a supplemental activation of 7.5%, no fragments resulting from the loss or transfer of the phosphate group are observed. This is advantageous as it will increase the reliability of the phosphopeptide analysis as there will be accurate assignment of the phosphorylated residue.
This experiment was carried out by varying the supplemental activation and the dispersion voltages, however there are many other variables that can be altered in the FAIMS device. Further possible work could be carried out, for example varying the electrode temperature in the FAIMS or altering the helium content of the carrier gas.
c8-80 c8
Acknowledgements
H.J.Cooper,
A.J.Creese, The Mass Spectrometry Lab at The University of Birmingham and the British Mass Spectrometry Society
[M+2H]2+-49 c7 z7• z8• z11 c11 [y13]2+ [z13•]2+ y5 z3• c4 z4• c6 z9• y9 1000 1200 y12 z12• 1400 1600 c8-80 c13 z13 c14 [M+2H]2+ c8
z10•
INTERNATIONAL LABMATE - MARCH 2013
Relative Abundance Intensity of peak
Relative Abundance
Relative Abundance
Relative Abundance
L L LL
L L L LL L
L L L L
L L LL L L
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