MALDI‐Mass Spectrometric Imaging of Endogenous Metabolites in Biological Systems


Matrix‐assisted laser desorption/ionisation (MALDI)‐mass spectrometric imaging (MSI) is a powerful, relatively new technology that allows for the detection of molecules directly from biological systems and also provides the spatial distribution for these analytes within the tissue sample. MSI has gained considerable popularity for the study of endogenous metabolites as metabolites are the end product of various biochemical processes, and represent a biological snapshot of an organism's biochemistry at a given time. Although the underlying concept is simple, factors such as choice of ionisation method, sample preparation, instrumentation and data analysis must be taken into account and tailored for successful applications of MSI, especially for the study of small molecules. MALDI‐MSI has been successfully applied to the study of endogenous metabolites in animal, plant, bacteria and fungal tissues and has provided new insights into biological functions and pathways, interspecies interactions and novel natural products.

Key Concepts:

  • A metabolic profile provides a snapshot of an organism's metabolism and underlying biochemistry at a given time.

  • MALDI‐MSI provides spatial distribution of endogenous or exogenous molecules in a sample.

  • Advantages of MALDI‐MSI include the ability to detect a broad mass range of compounds, the ability to detect multiple analytes in a single experiment without the need for labels or prior knowledge of the analytes and the preservation of biologically relevant spatial information.

  • The study of small molecules with MALDI‐MSI presents unique challenges due to matrix interference and potential analyte delocalisation.

  • MALDI‐MSI has been extensively used to study metabolites and other compounds in animal tissue and has recently been applied to the study of small molecules in plant, bacteria and fungi.

Keywords: MALDI; mass spectrometry; imaging; metabolites; metabolomics; biological tissues; plants; bacteria; agar

Figure 1.

MALDI‐MSI workflow. Samples are prepared by sectioning tissue samples and mounting them on a glass slide or MALDI target plate followed by application of matrix. MSI uses a computer‐controlled X‐Y stage, which holds the sample, and allows for the laser to raster across the sample and mass spectra to be collected at every predefined raster point. After completing the 2D raster, an ion image can be created using software to select a single mass from the spectrum and the software will display the relative abundance of that ion as a heat map of signal intensity at each raster point.

Figure 2.

The result of MSI analysis of testis in negative‐ion mode. Hematoxylin and eosin staining results and ion images of 14 representative peaks. Scale bar is 100 μm. Reproduced with permission from Goto‐Inoue et al. ().

Figure 3.

Comparison of the metabolite distribution in nitrogen‐fixing and non‐fixing Medicago truncatula nodules. (a) Nodules on a wild‐type (WT) Medicago plant inoculated with the WT strain of rhizobia (WT/WT). (b) Nodules on a WT plant inoculated with the fixJ mutant of rhizobia (WT/fixJ). (c) Nodules on a Medicago dnf1–1 mutant inoculated with the WT strain of rhizobia (dnf1/WT). (d) Nodules on a Medicago dnf1–1 mutant inoculated with the fixJ mutant of rhizobia (dnf1/fixJ). (e) Nitrogen fixation activity. Each bar corresponds to the mean value of acetylene reduction for eight plants assayed 3 weeks after inoculation. Error bars indicate standard error (SE). (f–i)Distribution of the haem moiety at m/z 616.18 in the four different nodule types: WT/WT (f), WT/fixJ (g), dnf1/WT (h) and dnf1/fixJ (i). The haem moiety was detected only in fixing nodules in (f). (j–m) Distribution of the metabolite formononetin MalGlc at m/z 517.13 on WT/WT (j), WT/fixJ (k), dnf1/WT (l) and dnf1/fixJ (m), showing great similarity in contrast to (f)–(i). Scale bars=1 mm. Reproduced with permission from Ye et al. ().

Figure 4.

MSI of multiple complex samples. (A) Los Angeles garden soil. (B) La Jolla Shores beach marine sediment. (a) Respective photographs of microbial communities. (b) Overlay of multiple ions observed within the microbial community. Reproduced with permission from Gonzalez et al. (). © Elsevier.

Figure 5.

MALDI‐MSI of the interaction between GA40 (bacteria) and Aspergillu fumigatus fungi (left) compared to interaction of GA40 versus Aspergillu niger fungi (right) including the controls (a) GA40 on the bottom and A. fumigatus on the top (b) A. fumigatus control (c) GA40 control (d) GA40 on the bottom and A. niger on the top (e) A. niger control (half of a colony was imaged) (f) GA40 control. Reproduced with permission from Moree et al. (). © Springer.



Araujo P, Ferreira MS, de Oliveira DN et al. (2014) Mass spectrometry imaging: an expeditious and powerful technique for fast in situ lignin assessment in eucalyptus. Analytical Chemistry 86(7): 3415–3419.

Bhandari DR, Shen T, Rompp A, Zorn H and Spengler B (2014) Analysis of cyathane‐type diterpenoids from Cyathus striatus and Hericium erinaceus by high‐resolution MALDI MS imaging. Analytical and Bioanalytical Chemistry 406(3): 695–704.

Bjarnholt N, Li B, D'Alvise J and Janfelt C (2014) Mass spectrometry imaging of plant metabolites – principles and possibilities. Natural Product Reports 31(6): 818–837.

Caprioli RM, Farmer TB and Gile J (1997) Molecular imaging of biological samples: localization of peptides and proteins using MALDI‐TOF MS. Analytical Chemistry 69(23): 4751–4760.

Chen R, Hui L, Sturm RM and Li L (2009) Three dimensional mapping of neuropeptides and lipids in crustacean brain by mass spectral imaging. Journal of the American Society for Mass Spectrometry 20(6): 1068–1077.

Chen S, Chen L, Wang J et al. (2012) 2,3,4,5‐Tetrakis(3′,4′‐dihydroxylphenyl)thiophene: a new matrix for the selective analysis of low molecular weight amines and direct determination of creatinine in urine by MALDI‐TOF MS. Analytical Chemistry 84(23): 10291–10297.

Cohen MZ (1987) A historical overview of the phenomenologic movement. Image: Journal of Nursing Scholarship 19(1): 31–34.

Crossman L, McHugh NA, Hsieh YS, Korfmacher WA and Chen JW (2006) Investigation of the profiling depth in matrix‐assisted laser desorption/ionization imaging mass spectrometry. Rapid Communications in Mass Spectrometry 20(2): 284–290.

Debois D, Ongena M, Cawoy H and De Pauw E (2013) MALDI‐FTICR MS imaging as a powerful tool to identify Paenibacillus antibiotics involved in the inhibition of plant pathogens. Journal of the American Society for Mass Spectrometry 24(8): 1202–1213.

DeKeyser SS, Kutz‐Naber KK, Schmidt JJ, Barrett‐Wilt GA and Li L (2007) Mass spectral imaging of neuropeptides in crustacean nervous tissue by MALDI TOF/TOF. Journal of Proteome Research 6: 1782–1791.

Fitzgerald JJD, Kunnath P and Walker AV (2010) Matrix‐enhanced secondary ion mass spectrometry (ME‐SIMS) using room temperature ionic liquid matrices. Analytical Chemistry 82(11): 4413–4419.

Gerlich M and Neumann S (2013) MetFusion: integration of compound identification strategies. Journal of Mass Spectrometry 48(3): 291–298.

Gonzalez DJ, Xu Y, Yang YL et al. (2012) Observing the invisible through imaging mass spectrometry, a window into the metabolic exchange patterns of microbes. Journal of Proteomics 75(16): 5069–5076.

Goto‐Inoue N, Hayasaka T, Zaima N and Setou M (2011) Imaging mass spectrometry reveals changes of metabolites distribution in mouse testis during testicular maturation. Surface and Interface Analysis 44: 749–754.

Guenther S, Rompp A, Kummer W and Spengler B (2011) AP‐MALDI imaging of neuropeptides in mouse pituitary gland with 5 μm spatial resolution and high mass accuracy. International Journal of Mass Spectrometry 305(2–3): 228–237.

Hankin JA, Barkley RM and Murphy RC (2007) Sublimation as a method of matrix application for mass spectrometric imaging. Journal of the American Society for Mass Spectrometry 18(9): 1646–1652.

Heuberger AL, Broeckling CD, Kirkpatrick KR and Prenni JE (2014) Application of nontargeted metabolite profiling to discover novel markers of quality traits in an advanced population of malting barley. Plant Biotechnology Journal 12(2): 147–160.

Jehl B, Bauer R, Dorge A and Rick R (1981) The use of propane‐isopentane mixtures for rapid freezing of biological specimens. Journal of Microscopy‐Oxford 123(September): 307–309.

Kallback P, Shariatgorji M, Nilsson A and Andre PE (2012) Novel mass spectrometry imaging software assisting labeled normalization and quantitation of drugs and neuropeptides directly in tissue sections. Journal of Proteomics 75(16): 4941–4951.

Kobayashi T, Nishiumi S, Ikeda A et al. (2013) A novel serum metabolomics‐based diagnostic approach to pancreatic cancer. Cancer Epidemiology Biomarkers & Prevention 22(4): 571–579.

Lemaire R, Tabet JC, Ducoroy P et al. (2006) Solid ionic matrixes for direct tissue analysis and MALDI imaging. Analytical Chemistry 78(3): 809–819.

Makarov A (2000) Electrostatic axially harmonic orbital trapping: a high‐performance technique of mass analysis. Analytical Chemistry 72(6): 1156–1162.

Moree WJ, Yang JY, Zhao X et al. (2013) Imaging mass spectrometry of a coral microbe interaction with fungi. Journal of Chemical Ecology 39(7): 1045–1054.

Pendyala G, Want EJ, Webb W, Siuzdak G and Fox HS (2007) Biomarkers for neuroAIDS: the widening scope of metabolomics. Journal of Neuroimmune Pharmacology 2(1): 72–80.

Robichaud G, Garrard KP, Barry JA and Muddiman DC (2013) MSiReader: an open‐source interface to view and analyze high resolving power MS imaging files on Matlab platform. Journal of the American Society for Mass Spectrometry 24(5): 718–721.

Schwartz SA, Reyzer ML and Caprioli RM (2003) Direct tissue analysis using matrix‐assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation. Journal of Mass Spectrometry 38(7): 699–708.

Scott AJ, Jones JW, Orschell CM et al. (2014) Mass spectrometry imaging enriches biomarker discovery approaches with candidate mapping. Health Physics 106(1): 120–128.

Shariatgorji M, Nilsson A, Goodwin R et al. (2013) MALDI‐MS Imaging and Quantitation of Primary Amine Neurotransmitters Dopamine, GABA and Glutamate Directly in Brain Tissue Sections. 2013 American Society for Mass Spectrometry Annual Conference. Minneapolis, MN.

Shrivas K, Hayasaka T, Sugiura Y and Setou M (2011) Method for simultaneous imaging of endogenous low molecular weight metabolites in mouse brain using TiO2 nanoparticles in nanoparticle‐assisted laser desorption/ionization‐imaging mass spectrometry. Analytical Chemistry 83(19): 7283–7289.

Shroff R, Rulisek L, Doubsky J and Svatos A (2009) Acid‐base‐driven matrix‐assisted mass spectrometry for targeted metabolomics. Proceedings of the National Academy of Sciences of the USA 106(25): 10092–10096.

Spegel P, Sharoyko VV, Goehring I et al. (2013) Time‐resolved metabolomics analysis of beta‐cells implicates the pentose phosphate pathway in the control of insulin release. Biochemical Journal 450(3): 595–605.

Thomas A, Charbonneau JL, Fournaise E and Chaurand P (2012) Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5‐diaminonapthalene deposition. Analytical Chemistry 84(4): 2048–2054.

Traxler MF, Watrous JD, Alexandrov T, Dorrestein PC and Kolter R (2013) Interspecies interactions stimulate diversification of the Streptomyces coelicolor secreted metabolome. mBio 4(4): e00459-13.

Verhaert PDEM, Pinkse MWH, Strupat K and Conaway MCP (2010) Imaging of similar mass neuropeptides in neuronal tissue by enhanced resolution MALDI MS with an ion trap‐orbitrap(™) hybrid instrument. Mass Spectrometry Imaging: Principles and Protocols 656: 433–449.

Wei R (2011) Metabolomics and its practical value in pharmaceutical industry. Current Drug Metabolism 12(4): 345–358.

West PR, Weir AM, Smith AM, Donlet ELR and Cezar GG (2010) Predicting human developmental toxicity of pharmaceuticals using human embryonic stem cells and metabolomics. Toxicology and Applied Pharmacology 247(1): 18–27.

Wolf S, Schmidt S, Muller‐Hannemann M and Neumann S (2010) In silico fragmentation for computer assisted identification of metabolite mass spectra. BMC Bioinformatics 11: 148.

Ye H, Gemperline E and Li LJ (2013a) A vision for better health: mass spectrometry imaging for clinical diagnostics. Clinica Chimica Acta 420: 11–22.

Ye H, Gemperline E, Venkateshwaran M et al. (2013b) MALDI mass spectrometry‐assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula‐Sinorhizobium meliloti symbiosis. Plant Journal 75(1): 130–145.

Zhou B, Wang JL and Ressom HW (2012) MetaboSearch: tool for mass‐based metabolite identification using multiple databases. Plos One 7(6): e40096.

Further Reading

Gemperline E, Chen B and Li L (2014) Challenges and recent advances in mass spectrometric imaging of neurotransmitters. Bioanalysis 6(4): 525–540.

Gemperline E and Li L (2014) MALDI‐mass spectrometric imaging for the investigation of metabolites in Medicago truncatula root nodules. Journal of Visualized Experiments (85): e51434.

Lee YJ, Perdian DC, Song ZH, Yeung ES and Nikolau BJ (2012) Use of mass spectrometry for imaging metabolites in plants. Plant Journal 70(1): 81–95.

Miura D, Fujimura Y and Wariishi H (2012) In situ metabolomic mass spectrometry imaging: recent advances and difficulties. Journal of Proteomics 75(16): 5052–5060.

Watrous JD and Dorrestein PC (2011) Imaging mass spectrometry in microbiology. Nature Reviews Microbiology 9(9): 683–694.

Yang JY, Phelan VV, Simkovsky R et al. (2012) Primer on agar‐based microbial imaging mass spectrometry. Journal of Bacteriology 194(22): 6023–6028.

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Gemperline, Erin, and Li, Lingjun(Aug 2014) MALDI‐Mass Spectrometric Imaging of Endogenous Metabolites in Biological Systems. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0023207]