Combining elemental and molecular mass spectrometry to study 3 types of biologically important compounds: DNA, phosphopeptides and anticancer drugs

Mass spectrometry was used to investigate three important biological molecules, deoxyribonucleic acid (DNA), phosphopeptides and oxaliplatin. The quantification of DNA is traditionally performed by UV spectroscopy; however the results can be affected greatly by the sample matrix. The method developed quantified phosphorus in digested calf thymus DNA and human DNA by high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). The method presented showed excellent baseline separation between all 4 DNA mono-nucleotides and 5 UMP. Column recoveries ranging from 95% to 99% for phosphorus resulted in a mass balance of 95% ± 0.5% for standard nucleotides, determined by LC-ICP-MS, compared to total DNA determined by flow injection coupled to ICP-MS (FI-ICP-MS). Protein phosphorylation and de-phosphorylation is one of the most common signalling pathways within cells, it is involved in regulating cellular processes, mediating enzyme inhibition, protein-protein recognition and protein degradation. A novel approach to the selective detection of phosphopeptides based on the incorporation of a metal tag, gallium N,N-biscarboxymethyl lysine (Ga-LysNTA), in solution before separation and detection by liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS) was developed. Linear ion trap electrospray ionisation mass spectrometry (ESI-MS) was employed to study the interaction of the gallium tag with platelet derived growth factor beta receptor (β-PDGF), a small phosphopeptide. In addition molecular modelling was used to investigate the energetically favoured structures of both the Ga-LysNTA material and the β-PDGF-Ga-LysNTA complex. The complexation of the Pt-based anti-cancer drug oxaliplatin (OxPt) with biological ligands other than DNA is believed to be a major cellular sink for the drug reducing its therapeutic potential and acting as a potential cause of toxicity. The role of the naturally abundant cytoplasmic dipeptide ligand β-alanyl-L-histidine dipeptide (carnosine) in OxPt detoxification was investigated. Various mass spectrometry techniques employing electrospray ionization and chip nanospray were employed to study the interaction of oxaliplatin with carnosine as well as two of its derivatives β-alanyl-N-methylhistidine (anserine) and N-acetylcarnosine (NAC). Evidence of complexation between OxPt and each of the three ligands examined is presented. Most species observed were unambiguously assigned and compared to their theoretical isotopic patterns

An ICP-MS, ESI-MS and molecular modelling investigation of homogeneous gallium affinity tagging (HMAT) of phosphopeptides

Protein phosphorylation and de-phosphorylation, provide one of the most common signalling pathways within cells, being involved in regulating cellular processes, mediating enzyme inhibition, protein-protein recognition and protein degradation. Compared with normal proteomics, phosphoproteomics poses some additional challenges requiring more initial separation and additional sensitivity to detect and quantify potentially ultra-low abundance species. In this work, the selective detection of phosphopeptides is described based on the incorporation of a metal tag, gallium-N,N-biscarboxymethyl lysine (Ga-LysNTA), in solution before separation and detection by liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS). Experimental and theoretical characterisation of the resulting Ga-phosphopeptide complex is presented based on linear ion trap electrospray ionisation mass spectrometry (ESI-MS), Fourier transform mass spectrometry (FT-MS) and molecular modelling data. Linear ion trap electrospray ionisation mass spectrometry (ESI-MS) was employed to study the interaction of the gallium tag with platelet derived growth factor beta receptor (β-PDGF), a small phosphopeptide. In addition high resolution Fourier transform mass spectrometry (FT-MS) was used for accurate mass determination and multistage tandem mass spectrometry of the gallium-β-PDGF complex identified the fragmentation pathway. Finally, molecular modelling was used to investigate the energetically favoured structures of both the Ga-LysNTA material and the β-PDGF-Ga-LysNTA complex

Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography-inductively coupled plasma-mass spectrometry

The determination of total deoxyribonucleic acid (DNA) concentration is of great importance in many biological and bio-medical analyses. The quantification of DNA is traditionally performed by UV spectroscopy; however the results can be affected greatly by the sample matrix. The proposed method quantifies phosphorus in digested calf thymus DNA and human DNA by high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). The method presented showed excellent baseline separation between all 4 DNA mono-nucleotides and 5’UMP. Column recoveries ranging from 95% to 99% for phosphorus resulted in a mass balance of 95% ± 0.5% for standard nucleotides, determined by LC-ICP-MS, compared to total DNA determined by flow injection coupled to ICP-MS (FI-ICP-MS). The ability of LC-ICPMS to act as an internal check that only DNA derived phosphorus was counted in the assay was demonstrated by establishing a mass balance between the total phosphorous signal from undigested DNA and that from the speciated DNA. The method for quantification was evaluated by analysis of NIST SRM 2372; a total speciated DNA recovery of 52.1 ng/μL, compared with an expected value of 53.6 ng/μL, was determined by external calibration. From repeat measurements a mass balance of 97% ± 0.5% for NIST DNA was achieved. The method limits of detection for individual nucleotides were determined between 0.8 to 1.7 μg L-1 (31P) for individual nucleotides by LC-ICP-MS, and 360 ng L-1 for 5’AMP by direct nebulisation

Oxaliplatin complexes with carnosine and its derivatives: in vitro cytotoxicity, mass spectrometric and computational studies with a focus on complex fragmentation

The complexation of the Pt-based anti-cancer drug oxaliplatin (OxPt) with biological ligands other than DNA is believed to be a major cellular sink for the drug reducing its therapeutic potential and acting as a potential cause of toxicity. In this paper, an in vitro study on hepatocellular carcinoma HepG2 cells suggests that the naturally abundant cytoplasmic dipeptide ligand β-alanyl-L-histidine dipeptide (carnosine) may inhibit the cytotoxic action of OxPt most likely through the formation of complexes that are less cytotoxic than OxPt alone. Evidence is provided to suggest that pre-exposure of HepG2 cells to elevated levels of carnosine appears to have a lasting effect on reducing the cytotoxicity of OxPt even after the removal of the carnosine. This effect, however, is shown to be under kinetic control as its magnitude was shown not to vary significantly with the level of carnosine exposure within the concentration range used in this study. Various mass spectrometry techniques employing electrospray ionization and chip nanospray were employed to study the interaction of oxaliplatin with carnosine as well as two of its derivatives being β-alanyl-N-methylhistidine (anserine) and N-Acetylcarnosine (NAC). Evidence of complexation between OxPt and each of the three ligands examined is presented. Most species observed were unambiguously assigned and compared to their theoretical isotopic patterns. Common fragmentation products due to the collisionally-activated protonated complexes of each of the ligands examined with OxPt, [M + OxPt + H]+ where M= carnosine, anserine or NAC were reported. Density functional calculations at B3LYP/LANL2DZ were used to obtain structural information and relative free energies of different isomers of the observed precursor [Carnosine + OxPt + H]+ both in the gas phase and in solution as well as to probe its fragmentation, highlighting plausible fragmentation mechanisms that account for all the experimental results.Data are presented to show several binding modes between electron rich sites such as N and O centers of carnosine and the Pt metal of OxPt. Calculations were also employed to obtain proton affinities and free energies of key reactions. The proton affinities of carnosine, Anserine and NAC at 298 K were calculated to be 254.4, 255.9 and 250.2 kcal mol-1 respectively. To the best of our knowledge the proton affinities of anserine and N-acetyl-carnosine are the first reported values in the literature