Mass spectrometry and metallomics: A general protocol to assess stability of metallodrug-protein adducts in bottom-up MS experiments.
The bottom-up mass spectrometry approach is today one of the best tools of Metallomics to characterize the binding of metal-based drugs to proteins. Yet, the stability of metal-protein coordination bonds along the whole process may be a critical issue. This led us to build up a general protocol to test metallodrug-protein adduct stability under the typical conditions of the filter-aided sample preparation (FASP)/bottom-up procedure, ranging from the analysis of solutions containing metal-protein adducts to tandem mass spectrometry experiments. More in detail, we identified nine critical situations, either during the sample manipulations or instrumental, as a potential source of metal-protein bond impairment when using FASP operative conditions and a nano high performance liquid chromatography-nanoelectrospray ionization-LTQ-Orbitrap (nanoLC-nanoESI-LTQ-Orbitrap) mass spectrometer system, equipped with a preconcentration/purification device. These are: 1) sample permanence in the ammonium bicarbonate buffer; 2) denaturation with urea; 3) reduction with dithiothreitol; 4) alkylation with iodoacetamide; 5) sample permanence in the loading mobile phase; 6) sample permanence in the elution mobile phase; 7) the nanoESI process; 8) the transfer of the adduct through ion transfer tube and tube lens; 9) collision induced dissociation in the ion trap. Accordingly, an ad hoc experimental protocol was developed and applied to the adducts formed between cytochrome c (Cyt c) and two different metallodrugs, i.e. cisplatin (cis-diamminedichloridoplatinum(II), CDDP) and RAPTA-C, a well-known ruthenium(II)-arene compound [Ru(η6-p-cymene)Cl2(pta)] (pta=1,3,5-triaza-7-phosphaadamantane), used here as models. Notably, Cyt c-CDDP adducts were stable through all the above conditions while Cyt c-RAPTA-C adducts turned out unstable in the ammonium bicarbonate buffer. This latter finding supports the need to perform a test-protocol of this kind when starting any extensive bottom-up MS investigation of protein-metallodrug systems.
Mass Spectrometry Center (CISM), University of Florence, via Ugo Schiff 6, 50019 Sesto Fiorentino, FI, Italy. Electronic address: elena.michelucci@unifi.it.
Elena
E
Michelucci
Mass Spectrometry Center (CISM), University of Florence, via Ugo Schiff 6, 50019 Sesto Fiorentino, FI, Italy.
Giuseppe
G
Pieraccini
Mass Spectrometry Center (CISM), University of Florence, via Ugo Schiff 6, 50019 Sesto Fiorentino, FI, Italy.
Gloriano
G
Moneti
Department of Chemistry and Industrial Chemistry, University of Pisa, via Moruzzi 13, 56124 Pisa, Italy.
Chiara
C
Gabbiani
MetMed, Department of Chemistry, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, FI, Italy.
Alessandro
A
Pratesi
MetMed, Department of Chemistry, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, FI, Italy.
Luigi
L
Messori
Binding site location; Bottom-up mass spectrometry; Cisplatin; Cytochrome c; Metallodrug-protein adducts; RAPTA-C