BOTTOM-UP MASS SPECTROMETRY PROTEOMICS STRATEGIES FOR THE IDENTIFICATION OF METALLODRUG
BINDING SITES ON PROTEINS: THE SEARCH FOR A GENERAL PROTOCOL TO ASSESS ADDUCT STABILITY
Elena Michelucci,a Giuseppe Pieraccini,a Gloriano Moneti,a Luigi Messori b
a Mass Spectrometry Center (CISM), University of Florence, via U. Schiff, 6, 50019 Sesto Fiorentino (FI), Italy
b Department of Chemistry “Ugo Schiff”, University of Florence, via della Lastruccia, 3, 50019 Sesto Fiorentino (FI), Italy
INTRODUCTION
REFERENCES
RESULTS AND DISCUSSION
Fig. 1. ESI-Orbitrap deconvoluted mass spectra; direct infusion of a 5 µM Mb solution.
CytC-cisPt adducts proved their stability toward all conditions listed above and so they showed to be ideal candidates for a binding site investigation using a bottom-up approach.
In our opinion, for cisPt it is not necessary to apply again the full protocol to test the complex with proteins different from CytC since, during the protocol application, the protein is denatured. Instead, it is strongly suggested to use it studying Pt-complexes different from cisPt or with other metals (Ru, Au).
Finally, experiments were carried out to evaluate Pt-protein and Pt-ligand stability in CID (point VIII). We used a simplified system consisting of three model peptides, each separately incubated with cisPt: MRFA (containing a Met), EHSG (containing a His) and GSH (glutathione, containing a Cys), being His (N-donor), Met and Cys
(S-donors) the main binding sites of Pt(II) on proteins7.
In each incubation mixture a Pt-peptide adduct ion was selected for CID experiments. The MS/MS spectra showed the stability of Pt-Met, Pt-His and Pt-Cys bonds (fig. 4a-b; data not shown for cisPt-GSH) while a partial loss of Pt ligands was observed (fig. 4b,
loss of NH3 and DMSO). In particular the study of this last kind of fragmentation gives
us important information to be used in the subsequent reworking of the bottom-up results through MaxQuant software.
1. Moreno-Gordaliza E. et al., Analyst, 2010, 135, 1288-1298.
2. Karas M., Bahr U., Dülcks T., Fresenius. J. Anal. Chem., 2000, 366, 669-676.
3. Jecklin M. C., Schauer S., Dumelin C. E., Zenobi R., J. Mol. Recognit., 2009, 22, 319-329. 4. Gabelica V. et al., Rapid Commun. Mass Spectrom., 2002, 16, 1723-1728.
5. Will J., Wolters D. A., Sheldrick W. S., ChemMedChem, 2008, 3, 1696-1707. 6. Ariza A. et al., J. Proteomics, 2012, 77, 504-520.
7. Gabbiani C. et al., J. Biol. Inorg. Chem., 2008, 13, 755-764. 17000 17200 17400 17600 Da 0 50 100 R el at iv e A bu n da nc e 17589.08 holo-Mb 17566.09 17632.04 17650.02 apo-Mb 16950.92 Na+, NH 4+, H2O adducts Na+, NH 4+, H2O adducts a) Solvent = H2O Capillary temperature = 220°C Tube lens voltage = 230V Base peak intensity = 1.1·104
17000 17200 17400 17600 Da 0 50 100 R el a tiv e A bu nd a nc e 17610.06 17629.03 17588.08 17648.01 17670.00 holo-Mb 17566.10 Na+, NH 4+, H2O adducts d) Solvent = H2O Capillary temperature = 177°C Tube lens voltage = 140V Base peak intensity = 8.0·102
17000 17200 17400 17600 Da 0 50 100 R el at iv e A bu n da nc e apo-Mb 16950.99 holo-Mb 17566.15
c) Solvent = H2O/CH3CN 1/1 with 0.1% HCOOH Capillary temperature = 220°C
Tube lens voltage = 230V Base peak intensity = 2.3·105
12400 12600 12800 Da 0 50 1000 50 1000 50 100 R el at iv e A bu nd an ce 0 50 1000 50 100 Pt mono-adducts Pt bis-adducts CytC 12358.31 t = 0 t = 48h t = 24h t = 72h t = 144h
Fig. 2. a) cisplatin. b) ESI-Orbitrap
deconvoluted mass spectrum of cisPt/CytC 3/1 solution after 10 days of incubation at 37°C in tetramethylammonium acetate buffer pH 6.8; direct infusion after dilution with H2O to a 5 µM CytC final concentration.
Fig. 3.
ESI-Orbitrap deconvoluted mass spectra; cisPt/CytC 3/1 solution after different incubation time at 37°C in tetramethylammonium acetate buffer pH 6.8; direct infusion after dilution with H2O to a 5 µM CytC final concentration.
Fig. 4. ESI-MS/MS spectra of a) [MRFA+Pt2+-H+]+ and b) {EHSG+[Pt(DMSO)(NH
3)]2+-H+}+.
Samples diluted in H2O/CH3CN 1/1 with 0.1% HCOOH to a final 50 M peptide concentration just before direct infusion. Fragmentation in IT (collision energy 30 arbitrary units), Orbitrap acquisition. cisPt a) 12400 12600 12800 Da 0 50 100 R e la tiv e A bu nd an ce CytC + [Pt(NH3)]2+ 12568.26 CytC 12358.29 CytC + [Pt(NH3)2]2+ 12587.28 CytC + 2[Pt(NH3)]2+ 12778.23 CytC + [Pt]2+ 12553.24 CytC + 2[Pt(NH3)2]2+ 12815.21 Pt mono-adducts Pt bis-adducts Na+, NH 4+, H2O adducts Na +, NH 4+, H2O adducts b) a) MS/MS spectrum of [MRFA+Pt2+-H+]+ M-R-F-A b1 b2 b3 y3 y2 y1 Pt2+ 200 300 400 500 600 700 m/z 0 50 100 R el at iv e A bu nd an ce 600.17 z=1 480.09 z=1 453.10 z=1 700.19 z=1 627.16 z=1 583.14 z=1 524.26 z=1 [b2++Pt2+-2H+]+ [b2++Pt2+-2H+-CO]+ [b3++Pt2+-2H+-CO]+ [b3++Pt2+-2H+-CO-NH 3]+ [MRFA+Pt2+-H+-NH 3]+ [MRFA+H]+ b) MS/MS spectrum of {EHSG+[Pt(DMSO)(NH3)]2+-H+}+ 200 300 400 500 600 700 m/z 0 50 100 R el at iv e A bu n da nc e 622.12 z=1 639.15 z=1 700.14 z=1 604.11 z=1 682.13 z=1 [EHSG+Pt2+-H+]+ [EHSG+Pt2+-H 2O-H+]+ {EHSG+[Pt(DMSO)]2+-H+}+ {EHSG+[Pt(NH3)]2+-H+}+ b1 b2 b3 y3 y2 y1 E-H-S-G [Pt(DMSO)(NH3)]2+ Bottom-up mass spectrometric approach (reduction, alkylation and enzymatic
digestion followed by MS/MS analysis) is one of the main methods used in proteomics to characterize the binding site of metal-based anticancer drugs on proteins. Nevertheless, in our opinion, the study of the stability of the metal fragment-protein coordination bond along the whole process has not received so far adequate
attention.1
Previous studies2 on myoglobin (Mb), that were confirmed in our mass spectrometry
facility, showed the instability of Fe-histidine coordination bond in electrospray (ESI) under certain preparative (pH, presence of an organic co-solvent) and instrumental (capillary temperature, tube lens voltage) conditions (fig. 1).
These findings convinced us to draw up a general protocol to test metal fragment– protein adduct stability under the typical conditions of the bottom-up approach, especially when the metal complex involved is not the well known and studied cisplatin (cisPt, fig. 2a) but a new metal complex containing other metals as Ru and Au.
We identified eight critical conditions as potential sources of metal-protein coordination bond impairment during the bottom-up process, using a LTQ-Orbitrap mass spectrometer:
I) sample permanence in ammonium bicarbonate2
II) dithiothreitol reduction1
III) iodoacetamide alkylation1
IV) permanence in loading mobile phases (CH3CN, 0.1% TFA)2
V) permanence in mobile phases (CH3CN, 0.1% HCOOH)2
VI) ESI process3,4
VII) transit through ion transfer tube (temperature) and tube lens (voltage)2
VIII) collision induced dissociation (CID) in ion trap (IT)5,6.
An experimental protocol was thus developed to assess the relevance of the above conditions on the stability of the metal-protein coordinative bond. First of all, the protocol was applied to the well known, model system cisPt-cytochrome C (CytC). CytC-cisPt adduct stability in conditions I), II), III), IV), V) and VII) was demonstrated by observing no variation in the relative intensities of the adduct peaks vs. the relative intensity of the unreacted CytC peak, before (fig. 2b) and after (data not shown) these treatments.
In ESI source, aspecific bond formation (false positives), as well as breaking of bonds present in solution (false negatives), could take place (point VI). The conservativeness of conventional ESI source conditions was proved by following the formation kinetic of cisPt-CytC adducts (fig. 3): the intensity of these peaks grows congruently with time and mutually unreacted CytC peak intensity decreases. A fortiori the softness of the nanoESI source, that will be used in bottom-up approach, will be ensured (absence of support gas and lower voltage applied).
CONCLUSIONS 17000 17200 17400 17600 Da 0 50 100 R e la tiv e A bu nd an ce apo-Mb 16950.95 16972.94 16993.92 holo-Mb 17566.12 17588.10 17016.90 17611.09 17632.07 Na+, NH 4+, H2O adducts Na +, NH 4+, H2O adducts b) Solvent = H2O/CH3CN 1/1 Capillary temperature = 220°C Tube lens voltage = 230V Base peak intensity = 2.6·105
