Material
All solvents were purchased from Thermo Fisher Scientific (Waltham, USA). The indium thin oxide (ITO)-coated glass slides were obtained from Bruker Daltonik (Bremen, Germany). The MALDI matrices as well as pure metabolite compounds were purchased from Sigma-Aldrich (Taufkirchen, Germany). The 10 μl tips and microloader tips were purchased from Eppendorf (Hamburg, Germany).
Tissue samples
Fresh frozen tumor tissues from 54 patients with predetermined IDH status were selected from the archive of the Department of Neuropathology, Heidelberg. Of those, 26 tumor tissues carried either an IDH1 or an IDH2 mutation, whereas the other 28 tumor tissues were IDH1/2 wildtype and served as negative test tissue (Additional file 1: Table S1). The series included 11 diffuse astrocytomas WHO grade II (DA), 4 anaplastic astrocytomas WHO grade III (AA), 7 oligodendroglioma (O), 3 anaplastic oligodendrogliomas (AO), 1 pilocytic astrocytomas WHO grade I (PA), 1 ganglioglioma WHO grade I (GG), 12 glioblastoma WHO grade IV (GBM), 13 schwannoma WHO grade I, and 1 non-small cell lung cancers (NSCLC). Of the IDH mutant DA, AA, O, AO and GBM 19 contained an IDH1R132H, 1 an IDH1R132C, 1 an IDH1R132G, 1 an IDH1R132S, 2 an IDH2R172K, 1 an IDH2R172S and 1 an IDH2R172M mutation.
Cases for analysis of the IDH-status via detection of 2HG were selected according to the following criteria: 1) knowledge of IDH-status, 2) tissue size sufficient for repeated analyses, 3) sufficient viable tumor tissue contained. For IDH wildtype samples, most tissues were selected from different brain tumor subtypes and one CNS tissue with reactive change. For IDH-mutant cases, gliomas with different IDH-mutations were collected. Examples for pre-characterization of tissues are shown in Fig. 1a-d. All samples were analyzed in an anonymous way. Informed consent has been provided in accordance with the local ethics committee.
Morphological analysis and immunohistochemistry
Prior to inclusion of samples, immunohistochemical staining for IDH1 R132H-mutant protein have been performed as described previously [7].
From archived tissue, frozen sections were cut to 4 μm with a Leica CM 3050 S cryostat (Leica Biosystems, Nussloch, Germany) in a defined orientation. H&E stainings from all frozen sections were analyzed microscopically to ensure the presence of vital tumor cells. Vital areas were marked on the slides for orientation in the MALDI-TOF-assay.
Detection of rare IDH mutations by sequencing
Prior to inclusion of samples, IDH1 exon 4 encompassing codon 132 and IDH2 exon 4 encompassing codon 172 have been subject to analysis by direct sequencing using an ABI 3100 DNA analyzer (Thermo Fisher Scientific, Waltham, USA) as previously described [13].
D-2HG detection by biochemical assay
The D-2HG assay has been described previously [3]. In brief, three 10 μm-thick slices were dissolved in 125 μl cell lysis buffer (150 mM NaCl, 0.1% NP-40, 50 mM Tris-HCl, pH 8.0) and subsequently treated with a deproteinization kit (Biovision, Mountain View, CA, USA). Supernatants were then collected and stored at − 20 °C. The total enzymatic reaction volume was 100 μl. Ten milliliters of assay solution were freshly prepared for each 96-well plate subjected to D-2HG assay. The assay solution contained 100 mM HEPES pH 8.0, 100 μM NAD+, 5 μM resazurin (Applichem, Darmstadt, Germany), 0.1 μg HGDH and 0.01 U/ml diaphorase (0.01 U/ml; MP Biomedical, Irvine, USA). Immediately before use, 25 μl sample volume was added to 75 μl of assay solution and incubated at room temperature for 30 min in black 96-well plates (Thermo Fisher Scientific, Waltham, USA) in the dark. Fluorometric detection was performed in triplicate with 25 μl deproteinized sample being analyzed in each reaction with excitation at 540 ± 10 nm and emission of 610 ± 10 nm (FLUOstar Omega, BMG Labtech, Offenburg, Germany).
Maleic anhydride proton sponge (MAPS) synthesis
MAPS was synthesized according to previously reported procedures [12, 24]: A solution of 1,8-Bis(dimethylamino)naphthalene (1.1 ml, 12 mmol – Sigma-Aldrich) in anhydrous THF (35 ml) was added to an orange solution of bromovaleric anhydride (5.0 g, 24 mmol – Sigma-Aldrich) in anhydrous THF (20 ml) under Argon at room temperature, immediately producing a deep red suspension. After 1 h, the suspension was concentrated in vacuo, redissolved in THF (60 ml) and filtered through paper. The filtrate was concentrated to give a purple-blackish crystalline solid (3.5 g, 95% yield).
1H NMR (400 MHz, CDCl3) δ: 7.90 (d, J = 8.5 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.87 (s, 1H), 6.85 (d, J = 8.1 Hz, 1H), 2.95 (s, 6H), 2.80 (s, 6H).
13C NMR (101 MHz, CDCl3) δ: 166.7, 165.4, 154.9, 151.8, 146.2, 136.3, 132.0, 128.2, 127.0, 121.6, 117.8, 116.2, 115.4, 112.5, 109.5, 43.4, 43.3.
HRMS (ESI) m/z: (M + H)+ calcd for C18H19N2O3: 311.1390; found: 311.1391.
Matrix solubilization and deposition on tissues
Four μm thick frozen sections were cut and thaw mounted onto ITO glass slides. Each slide contained both IDH wildtype and IDH-mutant sections. Brain tumor sections were dried at room temperature for 1 min.
Dihydroxybenzoic acid (DHB) was dissolved in a mixture of ACN/aqTFA 0.1% 7:3 at a concentration of 14 mg/ml. 9-amino acridine (9-AA) was dissolved in a mixture of MeOH/H2O 7:3 at a concentration of 10 mg/ml. 1,5-diaminonaphtalene (1,5-DAN) was dissolved in a mixture of ACN/aqTFA 0.1% 7:3 at a concentration of 6 mg/ml.
Different solvent mixtures were tested for the solubilization of 5 mg/ml of MAPS: ACN/aqTFA 0.1% 7:3, ACN/aqTFA 0.1% 9:1 and ACN/Chloroform 9:1.
The solutions were manually deposited on top of the regions of interest of the tissues, using a micropipette and 0.5–10 μl classical tips or microloader tips (Eppendorf, Wesseling-Berzdorf, Germany).
2HG profiling in tissues
Detection of 2HG in tissues was performed using the Rapiflex MALDI-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) which is equipped with a smartbeam laser (Nd:YAG 355 nm) operating at 10,000 Hz. The laser was set in MS dried droplet. MALDI analyses were operated in the reflector negative mode in order to detect the [M-H]- species of 2HG at m/z 147. The following settings were used: mass range analyzed: m/z 0–740, ions source 1 voltage: 19.87 kV, PIE: 2.417 kV, lens: 11.672, reflector 1: 20.835 kV, reflector 2: 1.01 kV, reflector 3: 8.58 kV, detector gain: 3135 V, sample rate 5GS/s, analog offset: 70.1 mV, global attenuator offset: 14%, laser intensity: 70%, movement on samples spot: off, matrix suppression: deflector. The calibration was made in negative mode using maleic acid (m/z 115.01), glutaric acid (m/z 131.04), alpha ketoglutarate (m/z 145.02), ascorbic acid (m/z 175,03) and isocitric acid (m/z 191.03).
MALDI imaging of 1,5-DAN spots
MALDI imaging was performed using a raster step of 50 μm. 5000 shots were acquired per spot and images dataset were constructed using flex imaging (Bruker Daltonik, Bremen, Germany).
Statistical methods
All statistical analysis was performed with Sigma Plot Version 13.0. The corresponding test used for the analysis is depicted in the figure legend.