Spatial Distribution of Endogenous Tissue Protease Activity in Gastric Carcinoma Mapped by MALDI Mass Spectrometry Imaging
2018; Elsevier BV; Volume: 18; Issue: 1 Linguagem: Inglês
10.1074/mcp.ra118.000980
ISSN1535-9484
AutoresKatrin Erich, Kevin Reinle, Torsten Müller, Bogdan Munteanu, Denis Abu Sammour, Isabel Hinsenkamp, Tobias Gutting, Elke Burgermeister, Peter Findeisen, Matthias Ebert, Jeroen Krijgsveld, Carsten Hopf,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoAberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue's endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity - with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies. Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue's endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity - with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies. Proteases control cell and tissue protein homeostasis (1Klein T. Eckhard U. Dufour A. Solis N. Overall C.M. Proteolytic cleavage-mechanisms, function, and "Omic" approaches for a near-ubiquitous posttranslational modification.Chem. Rev. 2018; 118: 1137-1168Crossref PubMed Scopus (112) Google Scholar). They influence cell proliferation, tissue morphogenesis and remodeling, and they are therefore associated with many pathological conditions including cancer. Cellular and secreted proteases play important roles during tumor initiation, growth, as well as metastasis, and their activity is not restricted to tumor cells, but affects tumor-surrounding tissue in favor of tumor expansion (2Olson O.C. Joyce J.A. Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response.Nat. Rev. Cancer. 2015; 15: 712-729Crossref PubMed Scopus (379) Google Scholar, 3Merchant N. Nagaraju G.P. Rajitha B. Lammata S. Jella K.K. Buchwald Z.S. Lakka S.S. Ali A.N. Matrix metalloproteinases: their functional role in lung cancer.Carcinogenesis. 2017; 38: 766-780Crossref PubMed Scopus (116) Google Scholar). Hence, they are key drug targets (4Drag M. Salvesen G.S. Emerging principles in protease-based drug discovery.Nat. Rev. Drug Discov. 2010; 9: 690-701Crossref PubMed Scopus (428) Google Scholar). In the past, various optical methods including (near infrared; NIR) fluorogenic and bioluminescence-based substrate- or activity reporter probes have been used to visualize protease activity in vitro, in cultured cells and in vivo (5Baruch A. Jeffery D.A. Bogyo M. Enzyme activity–it's all about image.Trends Cell Biol. 2004; 14: 29-35Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 6Edgington L.E. Verdoes M. Bogyo M. Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes.Curr. Opin. Chem. Biol. 2011; 15: 798-805Crossref PubMed Scopus (139) Google Scholar). Analytical probes typically enable fluorescence quenching, fluorescence resonance energy transfer (FRET) 1The abbreviations used are:FRETfluorescence resonance energy transferABCammonium bicarbonateCANacetonitrileAEBSF4-(2-aminoethyl)benzenesulfonyl fluoride hydrochlorideBCAbicinchoninic acid assayBSAbovine serum albuminCAAchloroacetamideDHB2,5-dihydroxxybenzoic acidEDTAethylenediaminetetraacetic acidFAformic acidFTICRfourier-transform ion cyclotron resonanceH&Ehematoxylin & eosinITOindium tin oxideLFQlabel-free quantificationMALDI MSImatrix-assisted laser desorption/ionization mass spectrometry imagingNIRnear-infraredPBSphosphate buffered salinePCAprincipal component analysisPIMprotease-inhibitor mixtureRTroom temperatureSDSsodium dodecylsulfateTCAtrichloroacetic acidTCEPtris(2-carboxyethyl)phosphineTFAtrifluoroacetic acidTPAAtissue protease activity assay., and other optical read-outs. Whereas several probes have been described for cell studies and in vivo molecular imaging (7Weissleder R. Molecular imaging in cancer.Science. 2006; 312: 1168-1171Crossref PubMed Scopus (985) Google Scholar), direct visualization and biochemical investigation of protease activity in tissue sections remains an underexplored field of research. In a notable exception, Withana et al. topically applied fluorescence-quenched activity-based probes to fresh-frozen tissue sections and visualized protease activity at cellular resolution (8Withana N.P. Garland M. Verdoes M. Ofori L.O. Segal E. Bogyo M. Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes.Nat. Protoc. 2016; 11: 184-191Crossref PubMed Scopus (43) Google Scholar). fluorescence resonance energy transfer ammonium bicarbonate acetonitrile 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride bicinchoninic acid assay bovine serum albumin chloroacetamide 2,5-dihydroxxybenzoic acid ethylenediaminetetraacetic acid formic acid fourier-transform ion cyclotron resonance hematoxylin & eosin indium tin oxide label-free quantification matrix-assisted laser desorption/ionization mass spectrometry imaging near-infrared phosphate buffered saline principal component analysis protease-inhibitor mixture room temperature sodium dodecylsulfate trichloroacetic acid tris(2-carboxyethyl)phosphine trifluoroacetic acid tissue protease activity assay. In this study, we utilize a different analytical approach, matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI), for spatially-resolved analysis of endogenous protease activity in fresh-frozen tissue. MALDI-MS imaging is label-free, and it rapidly localizes biomolecules without prior knowledge of their presence. Sample preparation for MALDI-MS imaging is comparatively fast and, hence, it is of increasing interest for clinical pathology (9Casadonte R. Caprioli R.M. Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry.Nat. Protocols. 2011; 6: 1695-1709Crossref PubMed Scopus (214) Google Scholar, 10Aichler M. Walch A. MALDI Imaging mass spectrometry: current frontiers and perspectives in pathology research and practice.Lab. Investigation. 2015; 95: 422-431Crossref PubMed Scopus (270) Google Scholar). MSI is frequently used for analysis of the in situ distribution of proteins (11Balluff B. Elsner M. Kowarsch A. Rauser S. Meding S. Schuhmacher C. Feith M. Herrmann K. Rocken C. Schmid R.M. Hofler H. Walch A. Ebert M.P. Classification of HER2/neu status in gastric cancer using a breast-cancer derived proteome classifier.J. Proteome Res. 2010; 9: 6317-6322Crossref PubMed Scopus (60) Google Scholar, 12Munteanu B. Meyer B. von Reitzenstein C. Burgermeister E. Bog S. Pahl A. Ebert M.P. Hopf C. Label-free in situ monitoring of histone deacetylase drug target engagement by matrix-assisted laser desorption ionization-mass spectrometry biotyping and imaging.Anal. Chem. 2014; 86: 4642-4647Crossref PubMed Scopus (54) Google Scholar), lipids (13Chaurand P. Cornett D.S. Angel P.M. Caprioli R.M. From whole-body sections down to cellular level, multiscale imaging of phospholipids by MALDI mass spectrometry.Mol. Cell. Proteomics. 2011; 10 (O110 004259)Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14Hinsenkamp I. Schulz S. Roscher M. Suhr A.M. Meyer B. Munteanu B. Fuchser J. Schoenberg S.O. Ebert M.P. Wangler B. Hopf C. Burgermeister E. Inhibition of rho-associated kinase 1/2 attenuates tumor growth in murine gastric cancer.Neoplasia. 2016; 18: 500-511Crossref PubMed Scopus (27) Google Scholar), drugs (14Hinsenkamp I. Schulz S. Roscher M. Suhr A.M. Meyer B. Munteanu B. Fuchser J. Schoenberg S.O. Ebert M.P. Wangler B. Hopf C. Burgermeister E. Inhibition of rho-associated kinase 1/2 attenuates tumor growth in murine gastric cancer.Neoplasia. 2016; 18: 500-511Crossref PubMed Scopus (27) Google Scholar), as well as peptides (15Cillero-Pastor B. Heeren R.M. Matrix-assisted laser desorption ionization mass spectrometry imaging for peptide and protein analyses: a critical review of on-tissue digestion.J. Proteome Res. 2014; 13: 325-335Crossref PubMed Scopus (83) Google Scholar), and N-glycans (16Drake R.R. Powers T.W. Jones E.E. Bruner E. Mehta A.S. Angel P.M. MALDI mass spectrometry imaging of N-linked glycans in cancer tissues.Adv. Cancer Res. 2017; 134: 85-116Crossref PubMed Scopus (90) Google Scholar, 17Heijs B. Holst S. Briaire-de Bruijn I.H. van Pelt G.W. de Ru A.H. van Veelen P.A. Drake R.R. Mehta A.S. Mesker W.E. Tollenaar R.A. Bovée J.V.M.G. Wuhrer M. McDonnell L.A. Multimodal mass spectrometry imaging of N-glycans and proteins from the same tissue section.Anal. Chem. 2016; 88: 7745-7753Crossref PubMed Scopus (69) Google Scholar). A recent study with a neuropeptide, dynorphin, spotted onto rat brain slices suggested that it may be possible to detect the levels of substrate and peptide bioconversion products simultaneously by MSI (18Bivehed E. Stromvall R. Bergquist J. Bakalkin G. Andersson M. Region-specific bioconversion of dynorphin neuropeptide detected by in situ histochemistry and MALDI imaging mass spectrometry.Peptides. 2017; 87: 20-27Crossref PubMed Scopus (18) Google Scholar). Here, we take this approach one step further and use spray-coating techniques for application of a protease substrate tracer, substance P, onto tissue for effective monitoring of endogenous enzyme activity and of the transient emergence of cleavage products over time at high spatial resolution. We utilize fast MALDI-TOF and high resolving power MALDI-Fourier Transform Ion Cyclotron Resonance (FTICR) MSI to visualize for the first time tissue protease activity in mouse gastric carcinoma using MALDI MSI. Substance P was purchased from BACHEM (Bubendorf, Switzerland). Protease inhibitor mix (PIM), trichloroacetic acid (TCA), Folin & Ciocalteu's phenol reagent, casein (from bovine milk), bovine serum albumin (BSA), pancreatin (from porcine pancreas), Tris(2-carboxyethyl)phosphine (TCEP) and chloroacetamide (CAA) were obtained from Sigma-Aldrich (Steinheim, Germany). Conductive indium tin oxide (ITO)-coated glass slides and peptide calibration standard II were from Bruker Daltonik (Bremen, Germany), MALDI matrix 2,5-dihyroxybenzoic acid (DHB) from Alfa Aesar (Karlsruhe, Germany), and trifluoroacetic acid (TFA) and ethanol were from Merck (Darmstadt, Germany). Acetonitrile (ACN) was obtained from Fisher Scientific (Waltham, MA), acetone, acetonitrile and methanol from VWR Chemicals (Fontenay-sous-Bois, France), and Mayer's Hematoxylin solution from Sigma-Aldrich. Eosin G Solution 0.5%, magnesium sulfate, sodium bicarbonate, hydrochloric acid and l-tyrosine were all purchased from Carl Roth (Karlsruhe, Germany). All solvents were MS grade. Triton-X-100, potassium hydrogenphosphate (K2HPO4) and sodium carbonate (Na2CO3) were from Merck. Sodium dodecylsulfate (SDS) was from Applichem (Darmstadt, Germany) and phosphate buffered saline (PBS) was from biowest (Darmstadt, Germany). Ammonium formiate was purchased from VWR (Darmstadt, Germany), ammonium bicarbonate (ABC) from Fluka Analytical (Munich, Germany). Trifluoroacetic acid (TFA) and formic acid (FA) was from Biosolve Chemicals (Dieuze, France). Sequencing grade modified trypsin was obtained from Promega (Madison, WI). Porcine organs were obtained from the local slaughterhouse. Kidney, spleen, pancreas, liver and muscle were prepared in blocks from about 2 × 2 cm, and immediately frozen. Transgenic CEA424-SV40 Tag C57BL/6 J mice with gastric carcinoma were described elsewhere (19Thompson J. Epting T. Schwarzkopf G. Singhofen A. Eades-Perner A.-M. van der Putten H. Zimmermann W. A transgenic mouse line that develops early-onset invasive gastric carcinoma provides a model for carcinoembryonic antigen-targeted tumor therapy.Int. J. Cancer. 2000; 86: 863-869Crossref PubMed Google Scholar). Animal studies were conducted in agreement with ethical guidelines of Heidelberg University and approved by government authorities (Az I-17/07). Stomachs of transgenic mice were removed and immediately frozen. All tissues were immediately frozen and stored at −80 °C until further use. For MALDI MSI analysis, frozen tissue was cut into 10 μm slices on a CM1950 cryostat (Leica Biosystems, Nussloch, Germany) at a temperature of −17 °C and thaw-mounted onto ITO-slides. In washing experiments, slices were rinsed with 2 × 1 ml 50 mm ammonium formiate (pH 7 and pH 3). The tissue was vacuum-dried for 15 min at 4 °C. To assess proteolytic in situ activity, one "digest" slide and one "no digest" control slide were prepared per tissue type. For assessment of time-dependent endogenous protease activity on porcine tissue (5 time-points x 5 organs x 2 conditions, i.e. "digest" and "no digest" for a total of 50 MSI data sets), 4 × 1 μl protease inhibitor mix (PIM; 1x) consisting of 2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 0.3 μm aprotinin, 116 μm bestatin hydrochloride, 14 μm E-64, 1 mm ethylenediaminetetraacetic acid (EDTA) and 1 μm leupeptin was applied to each tissue. Additionally, 1 × 1 μl ddH2O was pipetted on each tissue as solvent control. For studies of inhibitor concentration-dependent protease activity on porcine tissue, a dilution series (0×, 0.1×, 0.25×, 0.5×, 1×, 2.5×, 10×) of PIM was prepared in H2O, and one μl of each dilution was spotted onto tissue at arbitrarily chosen positions (n = 2). Slides were kept on ice at all times. In experiments using mouse tumor samples, an additional tissue slice was covered with PIM, using the SunCollect (pump system, SunChrom, Friedrichsdorf) and the following conditions: 3 × 15 μl/min, Y-distance 25.5 mm, Speed in X = Low 4, Speed in Y = Medium 1. The final amount of PIM on tissue was 0.2 μl/mm2. After drying under vacuum for 3 min at room temperature (RT), porcine or mouse tissues on the "digest" slide (Fig. 1) were covered with the tracer protease substrate substance P (20 pmol/μl in ddH2O) using the SunCollect spray system as described for PIM above for a coating with 3.5 pmol/mm2 substance P. During the spray process, the slide was kept on a cool pad. After spraying, the slide was dried in desiccator for 3 min at RT. Both slides ("digest" and "no digest") were incubated simultaneously at 37 °C and 95% humidity in the SunDigest device (SunChrom) for 15 min, 30 min, 45 min, 60 min, 120 min or 360 min in time-course experiments and for 60 min in inhibitor-concentration-dependence experiments (porcine tissues). Mouse tissue was incubated for 30 min at 37 °C instead, as protease activity was apparently higher in snap-frozen mouse tissue than in slaughterhouse tissue, but otherwise treated as porcine tissue. Homogeneity of the substance P coating on "no digest" slides was evaluated, and experiment with inhomogeneous coatings were rejected. MALDI-TOF MSI with DHB matrix was performed as outlined (14Hinsenkamp I. Schulz S. Roscher M. Suhr A.M. Meyer B. Munteanu B. Fuchser J. Schoenberg S.O. Ebert M.P. Wangler B. Hopf C. Burgermeister E. Inhibition of rho-associated kinase 1/2 attenuates tumor growth in murine gastric cancer.Neoplasia. 2016; 18: 500-511Crossref PubMed Scopus (27) Google Scholar). Porcine tissues were analyzed using an ultrafleXtreme MALDI-TOF/TOF instrument (Bruker Daltonik) in reflector positive mode and a mass range of m/z 500–2500. Tissues were measured at a lateral resolution of 200 μm, summing up 300 shots per pixel. External quadratic calibration using peptide standard II was performed for all MS instruments. Data was acquired and processed using flexControl V3.4 and flexImaging V4.1 software (Bruker Daltonik). For mouse samples, a rapifleX MALDI-TOF instrument (and flexImaging V5.1 (Bruker Daltonik) was used at a lateral resolution of 50 μm (20 μm for the replicate experiment), summing up 400 shots per pixel. After MALDI-TOF data acquisition, the same slides were analyzed using a 7T solariX XR MALDI FT-ICR (Bruker Daltonik). For imaging, the instrument was used in positive ion mode in an m/z range of m/z 100–3000 with a raster width of 20 μm and 15 laser shots/pixel. The following parameters were used: Ion transfer (Funnel 1 150 V, Skimmer 1 15 V, Funnel RF Amplitude 70 Vpp); Octopole (Frequency 5 MHz, RF Amplitude 350 Vpp); Collision Cell (RF Frequency 2 MHz, RF Amplitude 1200 Vpp); Transfer Optics (Time of Flight 1.5 ms, Frequency 4 MHz, FR Amplitude 350 Vpp); Quadrupole (Q1 Mass 350 m/z); Excitation Mode (Sweep Excitation, Sweep Step time 15 μs). Spectra of mouse tumor stomach were recorded using 512k (FID 0.2447 s) data point transient, corresponding to an estimated resolving power of 33,000 at m/z 400 resulting in a manageable data size of 22 GB. The average detected mass resolution at m/z 1347.73 was 23,000. Internal calibration was done using a lock mass of m/z 780.551 [PC(34:2)+Na]+. Data acquisition was performed using ftmsControl (Bruker Daltonik); ion images were obtained from flexImaging 4.1. For verification of substance P and its peptide products, CID fragmentation was used. Data analysis was performed using Biotools 3.2 and Sequence Editor 3.2 software (Bruker Daltonik). Data is available via ProteomeXchange with identifier PXD011104. To export mean intensities, we manually drew regions-of-interest (ROI) on tissue around spots with inhibitor (+inhibitor) or without inhibitor (-inhibitor) on "digest" and "no digest" control slides, respectively. Mean spectra of those ROIs were exported into mMass (V5.5.0 (20Strohalm M. Kavan D. Novak P. Volny M. Havlicek V. mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data.Anal. Chem. 2010; 82: 4648-4651Crossref PubMed Scopus (569) Google Scholar)) for baseline-subtraction and peak picking (S/N > 5). Intensities of the m/z-values of interest were exported into Excel (Microsoft). Nonlinear regression analysis was performed in Prism 5.0 software (Graphpad Software). For statistical analysis of the mouse tumor tissue, TOF data sets were converted into imzML file format (21Schramm T. Hester A. Klinkert I. Both J.P. Heeren R.M. Brunelle A. Laprevote O. Desbenoit N. Robbe M.F. Stoeckli M. Spengler B. Rompp A. imzML–a common data format for the flexible exchange and processing of mass spectrometry imaging data.J. Proteomics. 2012; 75: 5106-5110Crossref PubMed Scopus (219) Google Scholar) using the flexImaging converter (resampled to 80,000 data points), whereas the centroided FT-ICR data sets were read directly by an in-house conversion tool. Subsequently, the data sets were imported into R 3.3.1 (R Foundation for Statistical Computing, Vienna, Austria) (22Team R. RStudio: integrated development for R. 2015; Google Scholar, 23Ihaka R. Gentleman R. R: A language for data analysis and graphics.J. Computational Graphical Statistics. 1996; 5: 299-314Google Scholar) using MALDIquant and MALDIquantForeign packages (24Gibb S. Strimmer K. MALDIquant: a versatile R package for the analysis of mass spectrometry data.Bioinformatics. 2012; 28: 2270-2271Crossref PubMed Scopus (358) Google Scholar). The segmentation was performed using spatially-aware shrunken centroid clustering (25Bemis K.D. Harry A. Eberlin L.S. Ferreira C.R. van de Ven S.M. Mallick P. Stolowitz M. Vitek O. Probabilistic segmentation of mass spectrometry (MS) images helps select important ions and characterize confidence in the resulting segments.Mol. Cell. Proteomics. 2016; 15: 1761-1772Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) (r = 2, k = 2, s = 3) as implemented in Cardina (26Bemis K.D. Harry A. Eberlin L.S. Ferreira C. van de Ven S.M. Mallick P. Stolowitz M. Vitek O. Cardinal: an R package for statistical analysis of mass spectrometry-based imaging experiments.Bioinformatics. 2015; 31: 2418-2420Crossref PubMed Scopus (144) Google Scholar). The variable r = 2 to distinguish between tumor and nontumor. The segmentation was performed based on the m/z segment of 798.54 ± 5 and 798.54 ± 0.05 for the TOF and FT-ICR data sets, respectively. In that mass range, the lipid PC(34:1)+K+ with m/z 798.54, known to be enriched in gastric tumor in this mouse model, is present (14Hinsenkamp I. Schulz S. Roscher M. Suhr A.M. Meyer B. Munteanu B. Fuchser J. Schoenberg S.O. Ebert M.P. Wangler B. Hopf C. Burgermeister E. Inhibition of rho-associated kinase 1/2 attenuates tumor growth in murine gastric cancer.Neoplasia. 2016; 18: 500-511Crossref PubMed Scopus (27) Google Scholar). No pre-processing was performed on the imported raw data. Intensities for Substance P and its peptide cleavage products were exported and grouped into tumorous- and nontumorous segment. For protein extraction, frozen porcine tissues were cut into 30 μm slices, and six slices were collected for one extraction. Extraction buffer (PBS/1% (w/v) Triton-X-100/0.05% (w/v) SDS) was added to a concentration of 500 mg/ml wet tissue weight. Samples underwent five cycles of sonication (5 min) and cooling on ice (2 min). Finally, the mixture was centrifuged (5 min, 10,000 × g), supernatant was collected and placed on ice. Protein quantification was done by Bradford assay. The protease activity assay was based on Cupp-Enyard, 2008 (27Cupp-Enyard C. Sigma's non-specific protease activity assay - casein as a substrate.J Vis Exp. 2008; (http://dx.doi.org/10.3791/899)Crossref PubMed Scopus (279) Google Scholar): Briefly, 500 μl casein (0.65% (w/v) in 50 mm K2HPO4) was placed on 37 °C for 5 min. Then 100 μl protein extract was added and incubated 10 min at 37 °C. The reaction was stopped by 500 μl 110 mm TCA and further incubation for 30 min at 37 °C. Protein precipitate was separated by centrifugation for 5 min at 10.000 × g. 500 μl 500 mm sodium carbonate and 100 μl Folin & Ciocalteau's phenol reagent were added to the supernatant and incubated 30 min at 37 °C. 1 mg/ml solutions of native and heat-denatured pancreatin were used as positive and negative controls, respectively. Tissue extraction buffer was used for dilution series of l-tyrosine (0.0055, 0.011, 0.022, 0.044, 0.055, 0.077 and 0.11 μmol; for calculation of released tyrosine) and for blanks. Absorbance was measured in 96-well plates at 660 nm (Electro Multiscan Spectrum, Thermo Fisher Scientific) for 10 min. Activity calculation was performed by the following equation: Acticity[unitml]=releasedtyrosine[μmol]×assay[mL]enzymevolume[ml]×incunationtime[min]×measurementvolume[ml](Eq. 1) For proteomics analysis, frozen TCEA-positive and WT mouse stomachs (n = 3) were cryosectioned into 6 × 60 μm slices and collected in vials (3 technical replicates for each WT sample, 2 technical replicates for all TCEA-positive samples). Additionally, one slice (10 μm) of each tissue was thaw-mounted onto ITO-slides, vacuum-dried, and used for MALDI MSI. Based on mass distribution of PC(34:1)+K+, the tumor location in TCEA-positive stomachs was determined and further slices were prepared. Tumor-regions and nontumor regions were collected in separate vials and stored at −80 °C until further processing (supplemental Fig. S38). Tissue sections were reconstituted in 200 μl lysis buffer (1% (w/v) SDS, 100 mm ABC pH 8.5). Each sample was probe-sonicated on-ice 2 × 15 s at 10% frequency and 2 × 15 s at 20% frequency with cooling on-ice between cycles. Low viscosity of the homogenate, indicative of sufficiently sheared DNA, determined the number of cycles. Samples were centrifuged at 15,000 × g, at 4 °C for 30 min, followed by protein quantification using a BCA assay. For each sample, 20 μg of protein were processed in a total volume of 10 μl (filled up with 100 mm ABC) and further combined with 3.3 μl of 4× reduction and alkylation buffer (4% SDS, 360 mm ABC, 40 mm TCEP, and 160 mm CAA). Samples were heated for 5 min at 95 °C to denature proteins and to reduce and alkylate cysteine residues. Cooled to RT, samples were further processed by SP3 sample clean-up procedure (28Hughes C.S. Foehr S. Garfield D.A. Furlong E.E. Steinmetz L.M. Krijgsveld J. Ultrasensitive proteome analysis using paramagnetic bead technology.Mol. Syst. Biol. 2014; 10: 757Crossref PubMed Scopus (512) Google Scholar, 29Sielaff M. Kuharev J. Bohn T. Hahlbrock J. Bopp T. Tenzer S. Distler U. Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range.J. Proteome Res. 2017; 16: 4060-4072Crossref PubMed Scopus (132) Google Scholar). In brief, 2 μl of pre-washed magnetic beads (20 μl each of Sera-Mag Speed Beads A and B (Fisher Scientific, Germany) washed 1× with 160 μl and 2× with 200 μl ddH2O, and re-suspended in 20 μl ddH2O for a final working concentration) as well as 15.2 μl 100% ACN were added to each sample to reach a final concentration of 50% ACN. Protein binding to beads was allowed for 18 min off a magnetic rack, followed by 2 min incubation on a magnetic rack to immobilize beads. The supernatant was removed and beads were washed 2× with 200 μl of 100% ethanol and 1× with 180 μl of 100% ACN. Beads were resuspended in 100 mm ABC and sonicated for 5 min in a water bath. Finally, sequencing-grade trypsin was added in an enzyme:protein ratio of 1:40 (5 μl of 0.1 μg/μl trypsin in ddH2O), and beads were pushed from the tube walls into the solution to ensure efficient digestion. Upon overnight incubation at 37 °C and 1000 rpm in a table-top thermomixer, samples were acidified to a final concentration of 0.5% FA and quickly vortexed. Peptides were recovered by immobilizing the beads on a magnetic rack and transferring the supernatant to new PCR tubes (28Hughes C.S. Foehr S. Garfield D.A. Furlong E.E. Steinmetz L.M. Krijgsveld J. Ultrasensitive proteome analysis using paramagnetic bead technology.Mol. Syst. Biol. 2014; 10: 757Crossref PubMed Scopus (512) Google Scholar). MS injection-ready samples were stored at −20 °C. Samples were diluted with Buffer A (0.1% FA in ddH2O) to enable the injection of 1.5 μg in 10 μl volume. Peptides were separated using the Easy NanoLC 1200 fitted with a trapping (Acclaim PepMap C18, 5 μm, 100 Å, 100 μm × 2 cm) and an analytical column (Acclaim PepMap RSLC C18, 2 μm, 100 Å, 75 μm × 50 cm). The outlet of the analytical column was coupled directly to a Q-Exactive HF Orbitrap (Thermo Fisher Scientific) mass spectrometer. Solvent A was ddH2O, 0.1% (v/v) FA and solvent B was 80% ACN in ddH2O, 0.1% (v/v) FA. The samples were loaded with a constant flow of solvent A at a maximum pressure of 800 bar, onto the trapping column. Peptides were eluted via the analytical column at a constant flow of 0.3 μl/min at 55 °C. During elution, the percentage of solvent B was increased linearly from 3 to 8% in 4 min, then from 8% to 10% in 2 min, then from 10% to 32% in a further 68 min, and then to 50% B in 12 min. Finally, the gradient was finished with 7 min at 100% solvent B, followed by 10 min 97% solvent A. Peptides were introduced into the mass spectrometer via a Pico-Tip Emitter 360 μm OD x 20 μm ID; 10 μm tip (New Objective) and a spray voltage of 2kV. The capillary temperature was set at 275 °C. Full scan MS spectra with mass range m/z 350 to 1500 were acquired in the Orbitrap with a resolution of 60,000 FWHM. The filling time was set to a maximum of 32 ms with an automatic gain control target of 3 × 106 ions. The top 20 most abundant ions per full scan were selected for an MS2 acquisition. The dynamic exclusion list was with a maximum retention period of 40 s. Isotopes, unassigned charges, and charges of 1, 5 to 8, and >8 were excluded. For MS2 scans, the resolution was set to 15,000 FWHM with an automatic gain control of 1 × 105 ions and maximum fill time of 50 ms. Raw files were processed using MaxQuant (version 1.5.1.2) (30Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9150) Google Scholar). For follow-up data analysis, raw file names as deposited in PRIDE can be matched with the ones in the search output folder by means of the supplemental file "Renaming_Scheme_MaxQuant_Erich.xlsx". The search was performed against the mouse Uniprot database (20180622_Uniprot_mus-musculus_canonical_reviewed; 16970 entries) using the Andromeda search engine with the following search criteria: enzyme was set to trypsin with up to 2 missed cleavages. Carbamidomethylation (C) and oxidation (M)/acetylation (protein N-term) were selected as a fixed and variable modifications, respectively (30Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range
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