Mass Spectrometry Evidence for Cisplatin As a Protein Cross-Linking Reagent

Abstract

Cisplatin is a potent anti-cancer drug, which functions by cross-linking adjacent DNA guanine residues. However within one day of injection, 65~98% of the platinum in the blood plasma is protein-bound. It is generally accepted that cisplatin binds to methionine and histidine residues, but what is often underappreciated is that platinum from cisplatin has a 2+ charge and can form up to four bonds. Thus, it has the potential to function as a cross-linker. In this report, the cross-linking ability of cisplatin is demonstrated by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with the use of standard peptides, the 16.8 kDa protein calmodulin (CaM), but was unsuccessful for the 64 kDa protein hemoglobin. The high resolution and mass accuracy of FTICR MS along with the high degree of fragmentation of large peptides afforded by collisionally activated dissociation (CAD) and electron capture dissociation (ECD) are shown to be a valuable means of characterizing cross-linking sites. Cisplatin is different from current cross-linking reagents by targeting new functional groups, thioethers, and imidazoles groups, which provides complementarity with existing cross-linkers. In addition, platinum(II) inherently has two positive charges which enhance the detection of cross-linked products. Higher charge states not only promote the detection of cross-linking products with less purification, but result in more comprehensive MS/MS fragmentation and can assist the assignment of modification sites. Moreover, the unique isotopic pattern of platinum flags cross-linking products and modification sites by mass spectrometry.

Use of Top-Down and Bottom-Up Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Mapping Calmodulin Sites Modified by Platinum Anticancer Drugs

“…The isotopic patterns of the [CaM+4Pt+nPt(NH 3 ) 2 +H] 13+ (n=2~6) ions were compared with the theoretical isotopic patterns, which fit with mean absolute deviation within 1.5 ppm range (see the inserts of Figure 1b′ and Table S-1). As previously observed, cisplatin preferentially binds to Met sites of CaM with all four cisplatin ligands displaced in low molar ratios of cisplatin-CaM mixtures; 35 herein, the maintenance of ligands, such as NH 3 , indicates that cisplatin might also bind to His, Asp or Glu residues in CaM sequence when increasing the concentration of cisplain in the cisplatin-CaM mixture. In addition, all the modified peaks shift to higher m/z region, namely, lower charge states, which supports the assumption that cisplatin binds to the carboxyl groups of Asp or Glu residues because deprotonation of a carboxyl group is needed before it binds to platinum(II).…”

“…To further localize the binding sites of platinum to CaM, CaM-cisplatin adducts at different molar ratios (1:1, 1:2, and 1:8) were trypsin-digested and analyzed by MS. In the 1:1 molar ratio sample of trypsin-digested CaM-cisplatin, in addition to the previously reported cross-linked species, 35 another platinum cross-linked species CaM(107-126)+Pt+CaM(142-148) as well as platinum modified CaM(38-74), CaM(107-126), and CaM(127-148) were also observed. The same Pt-modified peaks were also observed in the CaM-cisplatin (1:2) sample; however, when the molar concentration of cisplatin is eight times higher than CaM, the intensities of all Pt-modified peaks dropped significantly, and most of them could be barely detected (Figure S-2).…”

“…26,34 Previously, ECD has been successfully applied to cisplatin cross-linked peptides and protein-CaM by Li et al . 35 Here, the top-down ECD mass spectrometric approach is extended to map the binding sites for platinum anti-cancer drugs on CaM.…”

Mass spectrometry as a powerful tool to study therapeutic metallodrugs speciation mechanisms: Current frontiers and perspectives

“…[78,79] A combination of different fragmentation techniques (CID and ECD) as well as ion mobility mass spectrometry (IM-MS) allowed the determination of the sulfur atom of the methionine residue of substance P as the favoured binding site of cisplatin, whereas the His residues of angiotensin II and bombesin appeared to be preferred. [78][79][80] Tandem mass spectrometry techniques have been used to characterize cisplatin interactions with were recorded and showed that, even though the interactions between the complexes and the DNA strands were non-covalent in nature, they were strong enough to be observed in the gas phase during the analysis, and confirmed that PPCs do significantly stabilise the duplex structure of the oligonucleotide strands.…”

A gain and bandwidth enhanced transimpedance preamplifier for Fourier-transform ion cyclotron resonance mass spectrometry

“…1 and 2) has become the instrument of choice for proteomics, 3 biological imaging, [4][5][6] and other research involving complex mixtures 7 or macromolecules. [8][9][10] Positioned within an ultra high vacuum chamber (<10 −9 mbar), an ion cyclotron resonance (ICR) cell is the main analyzing component of FT-ICR MS. The ICR cell detects ion motion in the magnetic field by electrostatic induction.…”