MALDI Linear Ion Trap Mass Spec
Irinotecan has strong antitumor activity against a variety of human tumors as the drug binds to and prevents dissociation of the DNA topoisomerase I complex, which is involved in DNA replication. Irinotecan inhibits enzyme activity and thus the DNA replication process.
For this study, FaDu tumor xenografts with high vascular regions were analyzed to investigate which regions of the tumor are accessible or inaccessible to irinotecan and establish the suitability of MALDI MS-based imaging for cancer therapy research. The researchers attempted to locate the known metabolites of irinotecan within the tumor. The metabolic pathway and major known metabolites are shown in Figure 1.
Nude mice (genetic mutant mice with an inhibited inmune system) with transplanted human FaDu head and neck tumors were treated with irinotecan, methylseleno cysteine (MSC), both drugs in combination, or not treated at all. Doses of MSC, 0.2 mg/mouse/day, were given during seven days. 100 mg/kg of irinotecan was then administered by intravenous injection to both MSC treated and untreated mice. Mice livers and tumors were then excised, flash frozen, and stored at -80°C prior to analysis.
The frozen tumors and livers were sectioned to 12 µm thick and thaw-mounted onto nonconductive glass slides. The slides were placed under vacuum for approximately 15 minutes. Five additions of 0.1 µL of 6-aza-2-thiothymine (ATT) matrix were spotted on top of the tissue or sprayed using a commercial airbrush. The matrix solvent was 70/30 v/v methanol/0.1% trifluoroeacteic acid.
A Thermo Scientific LTQ XL mass spectrometer from Thermo Fisher Scientific coupled to a MALDI source was used for imaging mass spectrometry, with data-acquisition software to raster the tissue in the X and Y directions. Thermo Scientific ImageQuest™ software was employed to provide visualization of the distribution of the drugs within the tissue.
A 60 Hz nitrogen laser with beam diameter of ~100 µm impinged directly on to the MALDI plate at a 30º angle. Ion activation techniques of collision-induced dissociation (CID) and pulsed-Q dissociation (PQD) were employed. PQD effectively lowers the low mass range in LTQ ion traps to m/z 50.
The MALDI fragmentation spectrum in Figure 2 shows all major irinotecan ion fragments as expected from electrospray ionization.
Results show that irinotecan metabolites previously only identified in human urine were present in all of the drug-treated samples from mice (irinotecan or combination irinotecan plus MSC) and none of the metabolites were detected in either the no-drug or the MSC-only control.
SN-38 (m/z 393), SN-38G (m/z 569), isobaric M1 and/or M2 (m/z 603), and the parent drug irinotecan (m/z 587) were found. The MS/MS of m/z 603 contained weak fragment ions at 518 (-85, loss of piperidine) and m/z 502 (-101, loss of oxidized piperidine), which implied that both M1 and M2 metabolites were present in tissue.
However, the MS3 of m/z 518, which would confirm the presence of M1, was weak and not conclusive. The peak at m/z 603 appeared to be mostly M2 because of the prominent m/z 559 peak (-CO2). MS3 data confirmed this. The NPC metabolite (m/z 519), an indication of in vivo metabolized loss of a terminal piperidine, was not observed directly, although loss of piperidine was evident through CID in the ion trap.
Imaging mass spectrometry enabled the detection of distributions of unmetabolized irinotecan in the liver and FaDu tumor. The parent drug was convincingly identified from the tissue, as were the metabolites SN-38 (active metabolite), SN-38-G and M2, by MS/MS and MS3 tandem mass spectrometry in positive ion mode.
The distribution of two metabolites (SN-38 and its glucuronide form) in FaDu tumors treated with irinotecan alone and irinotecan plus MSC is shown in Figure 3. This second drug has been shown to increase irinotecan efficacy against tumors and is more effective in highly vascularized tumors such as FaDu than in avascular tumors.
Figure 3 clearly shows that m/z 349 (a fragment ion of SN-38 and SN-38-G) is more uniformly distributed in FaDu tumors that have been treated with both MSC and irinotecan than in those treated with irinotecan alone, where the analyte appears clumped in regions.
These results were consistent in three different tissue samples, two acquired at 100 µm resolution (MS/MS and MS3 data) and one at 50 µm resolution (MS/MS only). This may indicate that coating variability during matrix application was not to blame. Results from the MS3 image were more specific and indicative of the metabolite, as controls showed some signal for MS/MS of m/z 393.
MALDI linear ion trap mass spectrometry was able to identify and confirm most of the known drug metabolites in drug-treated tumor samples through MS/MS and MS3. The MALDI linear ion trap mass spectrometer enables isolation of the analyte of interest, accumulating ions in the ion trap and detecting all ions trapped through radial ejection and use of two electron multiplier detectors.
This technique can also generate a 2-D image, enabling the precise localization of specific analytes in the sample. When compared with TOF MS or MS2 detection, MALDI enabled linear ion trap offers MS3 fragmentation, an essential technique for tissue imaging, filtering interfering, and isobaric species that are part of the baseline chemical noise and might be isolated during MS/MS.