CHAPTER 3: Facile sampling of sweat combined with direct analysis of
3.3.7 The influence of sampling time
Figure 3.11. Analysis of a sweat sample on plaster A, following incubation of the plaster with skin of a volunteer, using nanoDESI-MS operated in the negative-ion mode. The solvent mixture used in this nanoDESI-MS experiment was pure methanol (LC-MS grade).
3.3.7 The influence of sampling time
In the final experiment, we evaluated the influence of sampling time on the signal-to-noise ratio. A series of samples were collected at varied sampling times: from 1 to 300 s. Interestingly, already after 10 s, a peak at the m/z 187 could be recorded (Figure 3.12).
The signal-to-noise ratio generally increased with the sampling time. The result confirms that sweat is secreted by skin all the time, and plaster patches can readily be used to collect samples of sweat from skin prior to the analysis by MS.
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Figure 3.12. Analysis of a sweat sample on plaster A, following incubation of the plaster with skin of a volunteer, using nanoDESI-MS operated in the negative-ion mode. The sampling time was varied (1 – 300 s). The solvent mixture used in this nanoDESI-MS experiment was composed of methanol and acetonitrile in the volume ratio 1:1, spiked with acetic acid (final concentration, 0.1%).
63 recorded by mass spectrometer. For example, creatine and urea could be detected in the dry sample deposits. We further optimized the way of collecting sweat samples as well as the key parameters of the nanoDESI-MS method in order to ensure satisfactory quality of mass spectra. The optimized parameters included: type of sampling material, nanoDESI solvent system, as well as the MS ion mode. It is noteworthy that the presented analytical method takes advantage of commercial plasters as simple sampling tools which enable facile collection of sweat samples. We have found that such plasters are also compatible with the nanoDESI-MS setup used as the main analytical platform. Signal of metabolite (N-acetyl-L-glutamine) could be detected by nanoDESI-MS following a very short sampling period (a few seconds). In future, one can apply this method to sweat analysis and further clinical diagnosis of sweat. The biggest problem observed during the development of this
Solvent Baseline Sensitivity
MeOH/Acetonitrile 0.1% acetic acid +++ +++
MeOH/ACN ++ +
MeOH + ++
Acetonitrile ++ +
64
method was a relatively high spectral background due to the contamination signals the solvent system or sampling plasters. To mitigrate this problem high quality solvents adoption should be used in future work. Although commercial plasters are a convenient facilitating tool, they may also contribute to spectral background. To further develop this method, one needs to find or develop more reliable materials that would not increase the spectral noise, and could concerntrate metabolites at the same time. In addition, one may consider using this simple sampling method with another ion source, for example direct analysis in real time (DART)72 and easy ambient sonic-spray ionization EASI73.
65
CHAPTER 4:
Conclusions
This reasearch work has led to the development of two methods for the analysis of metabolites in different kinds of biological samples by mass spectrometry. In the first study, we have developed a protocol for the analysis of small metazoan samples by matrix-assisted laser desorption/ionization mass spectrometry. The study has shown the feasibility of isotopic labeling of fruit flies with the purpose of pursuing metabolic effects of the circadian clock and environmental cues by mass spectrometry. The results also reveal a substantial metabolic inertia of the circadian clock: once adapted to the day/night cycle, the incorporation of 13C to UDP-Glc was not significantly altered by acute perturbation of the illumination cycle. We believe the method may also help to explore other biochemical phenomena in fruit fly as well as other metazoan species, which are commonly studied as model biological organisms. In the second part of this work, we have proposed application of adhesive plasters as sampling tools for sweat and subsequent analysis of such samples by nanospray desorption electrospray ionization. Following a short contact of plaster with skin (a few seconds), we could record one peak corresponding to a metabolite. The development of new sampling materials, which could reduce ion supperssion and enhance sensitivity, is needed. If successful, in future, the method may find applications in analysis and doping control. Overall, the work shows a broad applicability of mass spectrometric platforms in the analysis of metabolites in biological
66
samples. We have successfully used mass spectrometry in the analysis of different kinds of samples (microscale biological specimens and sweat). This confirms that, mass spectrometry can play an important role in future discoveries in bioscience.
67 Metabolite profiling for plant functional genomics. Nat Biotechnol 2000, 18, 1157-1161.
4. Oldiges, M.; Kunze, M.; Degenring, D.; Sprenger, G. A.; Takors, R., Stimulation, monitoring, and analysis of pathway dynamics by metabolic profiling in the aromatic amino acid pathway. Biotechnol Progr 2004, 20 (6), 1623-1633.
5. Fiehn, O.; Kopka, J.; Trethewey, R. N.; Willmitzer, L., Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry. Anal Chem 2000, 72, 3573-3580.
6. Villas-Boas, S. G.; Hojer-Pedersen, J.; Akesson, M.; Smedsgaard, J.; Nielsen, J., Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast 2005, 22, 1155-1169.
7. Villas-Boas, S. G.; Mas, S.; Akesson, M.; Smedsgaard, J.; Nielsen, J., Mass spectrometry in metabolome analysis. Mass Spectrom Rev 2005, 2, 613-646.
8. Nielsen, J.; Oliver, S., The next wave in metabolome analysis. Trends Biotechnol 2005, 23, 544-546.
9. Mungur, R.; Glass, A. D. M.; Goodenow, D. B.; Lightfoot, D. A., Metabolite fingerprinting in transgenic Nicotiana tabacum altered by the Escherichia coli glutamate dehydrogenase gene. J Biomed Biotechnol 2005, 2, 198-214.
10. Allen, J.; Davey, H. M.; Broadhurst, D.; Heald, J. K.; Rowland, J. J.; Oliver, S. G.; Kell, D.
B., High-throughput classification of yeast mutants for functional genomics using metabolic footprinting. Nat Biotechnol 2003, 21, 692-696.
11. Fuzfai, Z.; Katona, Z. F.; Kovacs, E.; Molnar-Perl, I., Simultaneous identification and quantification of the sugar, sugar alcohol, and carboxylic acid contents of sour cherry, apple, and ber fruits, as their trimethylsilyl derivatives, by gas chromatography-mass spectrometry. J Agr Food Chem 2004, 52, 7444-7452.
12. Buchholz, A.; Takors, R.; Wandrey, C., Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques. Anal Biochem 2001, 295, 129-137.
68
13. Iwatani, S.; Van Dien, S.; Shimbo, K.; Kubota, K.; Kageyama, N.; Iwahata, D.; Miyano, H.;
Hirayama, K.; Usuda, Y.; Shimizu, K.; Matsui, K., Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS. J Biotechnol 2007, 128, 93-111.
14. Noh, K.; Gronke, K.; Luo, B.; Takors, R.; Oldiges, M.; Wiechert, W., Metabolic flux analysis at ultra short time scale: Isotopically non-stationary C-13 labeling experiments. J Biotechnol 2007, 129, 249-267.
15. Karas, M.; Hillenkamp, F., Laser Desorption Ionization of proteins with molecular masses exceeding 10000 daltons. Anal Chem 1988, 60, 2299-2301.
16. Zenobi, R.; Knochenmuss, R., Ion formation in MALDI mass spectrometry. Mass Spectrom Rev 1998, 17, 337-366.
17. Knochenmuss, R.; Zenobi, R., MALDI ionization: The role of in-plume processes. Chem Rev 2003, 103, 441-452.
18. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., electrospray ionization for mass-spectrometry of large biomolecules. Science 1989, 246, 64-71.
19. Wollnik, H., Time-of-Flight Mass Analyzers. Mass Spectrom Rev 1993, 12, 89-114.
20. Imrie, D. C.; Pentney, J. M.; Cottrell, J. S., A Faraday cup detector for high-mass ions in matrix-assisted laser desorption/ionization Time-of-Flight mass-spectrometry. Rapid Commun Mass Spectrom 1995, 9, 1293-1296.
21. Loboda, A. V.; Krutchinsky, A. N.; Bromirski, M.; Ens, W.; Standing, K. G., A tandem quadrupole/time-of-flight mass spectrometer with a matrix-assisted laser desorption/ionization source: design and performance. Rapid Commun Mass Spectrom 2000, 14, 1047-1057.
22. Paradisi, C.; Todd, J. F. J.; Traldi, P.; Vettori, U., Boundary effects and collisional activation in a quadrupole ion trap. Org Mass Spectrom 1992, 27, 251-254.
23. Stafford, G. C.; Kelley, P. E.; Syka, J. E. P.; Reynolds, W. E.; Todd, J. F. J., Recent behaviour to genes - First in the Cycles Review Series. Embo Rep 2005, 6, 930-935.
26. Waterhouse, J.; Reilly, T.; Atkinson, G.; Edwards, B., Jet lag: trends and coping strategies.
Lancet 2007, 369, 1117-1129.
69
27. Froy, O., Metabolism and circadian rhythms-implications for obesity. Endocr Rev 2010, 31, 1-24.
28. O'Neill, J. S.; Reddy, A. B., Circadian clocks in human red blood cells. Nature 2011, 469, 498-507.
29. Bae, K.; Edery, I., Regulating a circadian clock's period, phase and amplitude by phosphorylation: Insights from Drosophila. J Biochem 2006, 140, 609-617.
30. Adams, M. D. et al., The genome sequence of Drosophila melanogaster. Science 2000, 287, 2185-2195.
31. Bier, E., Drosophila, the golden bug, emerges as a tool for human genetics. Nat Rev Genet 2005, 6, 9-23.
32. Shaw, P. J.; Cirelli, C.; Greenspan, R. J.; Tononi, G., Correlates of sleep and waking in Drosophila melanogaster. Science 2000, 287, 1834-1837.
33. Sehgal, A.; Mignot, E., Genetics of sleep and sleep disorders. Cell 2011, 146, 194-207.
34. Vanin, S.; Bhutani, S.; Montelli, S.; Menegazzi, P.; Green, E. W.; Pegoraro, M.; Sandrelli, F.; Costa, R.; Kyriacou, C. P., Unexpected features of Drosophila circadian behavioural rhythms under natural conditions. Nature 2012, 484, 371-U108.
35. Rubakhin, S. S.; Romanova, E. V.; Nemes, P.; Sweedler, J. V., Profiling metabolites and peptides in single cells. Nat Methods 2011, 8, S20-S29.
36. Svatos, A., Single-cell metabolomics comes of age: new developments in mass spectrometry profiling and imaging. Anal Chem 2011, 83, 5037-5044.
37. Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T., Matrix-assisted laser desorption ionization mass-spectrometry of biopolymers. Anal Chem 1991, 63, A1193-A1202.
38. Urban, P. L.; Schmidt, A. M.; Fagerer, S. R.; Amantonico, A.; Ibanez, A.; Jefimovs, K.;
Heinemann, M.; Zenobi, R., Carbon-13 labelling strategy for studying the ATP metabolism in individual yeast cells by micro-arrays for mass spectrometry. Mol Biosyst 2011, 7, 2837-2840.
39. Hu, J. B.; Chen, Y. C.; Urban, P. L., On-target labeling of intracellular metabolites combined with chemical mapping of individual hyphae revealing cytoplasmic relocation of isotopologues. Anal Chem 2012, 84, 5110-5116.
40. Gouw, J. W.; Pinkse, M. W. H.; Vos, H. R.; Moshkin, Y.; Verrijzer, C. P.; Heck, A. J. R.;
Krijgsveld, J., In Vivo stable isotope labeling of fruit flies reveals post-transcriptional regulation in the maternal-to-zygotic transition. Mol Cell Proteomics 2009, 8, 1566-1578.
70
41. Prasad, M.; Jang, A. C. C.; Starz-Gaiano, M.; Melani, M.; Montell, D. J., A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging. Nat Protoc 2007, 2, 2467-2473.
42. Rabinowitz, J. D.; Kimball, E., Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal Chem 2007, 79, 6167-6173.
43. Edwards, J. L.; Kennedy, R. T., Metabolomic analysis of eukaryotic tissue and prokaryotes using negative mode MALDI time-of-flight mass spectrometry. Anal Chem 2005, 77, 2201-2209.
44. Tsuboi, K. K.; Fukunaga, K.; Petricciani, J. C., Purification and specific kinetic properties of erythrocyte uridine diphosphate glucose pyrophosphorylase. The Journal of biological chemistry 1969, 244, 1008-15.
45. Parker, C. G.; Fessler, L. I.; Nelson, R. E.; Fessler, J. H., Drosophila Udp-glucose-glycoprotein glucosyltransferase ‒ Sequence and Characterization of an enzyme that distinguishes between denatured and native proteins. Embo J 1995, 14, 1294-1303.
46. Sezgin, E.; Duvernell, D. D.; Matzkin, L. M.; Duan, Y. H.; Zhu, C. T.; Verrelli, B. C.;
Eanes, W. F., Single-locus latitudinal clines and their relationship to temperate adaptation in metabolic genes and derived alleles in Drosophila melanogaster.
Genetics 2004, 168, 923-931.
47. Stoleru, D.; Nawathean, P.; Fernandez, M. D. L. P.; Menet, J. S.; Ceriani, M. F.; Rosbash, M., The Drosophila circadian network is a seasonal timer. Cell 2007, 129, 207-219.
48. Peschel, N.; Helfrich-Forster, C., Setting the clock - by nature: Circadian rhythm in the fruitfly Drosophila melanogaster. Febs Lett 2011, 585, 1435-1442.
49. Rogulja, D.; Young, M. W., Control of sleep by cyclin A and its regulator. Science 2012, 335, 1617-1621.
50. Rothenfluh, A.; Abodeely, M.; Price, J. L.; Young, M. W., Isolation and analysis of six timeless alleles that cause short- or long-period circadian rhythms in Drosophila.
Genetics 2000, 156, 665-675.
51. Grant, S. C.; Aiken, N. R.; Plant, H. D.; Gibbs, S.; Mareci, T. H.; Webb, A. G.; Blackband, S. J., NMR spectroscopy of single neurons. Magnet Reson Med 2000, 44, 19-22.
52. Kiessling, S.; Eichele, G.; Oster, H., Adrenal glucocorticoids have a key role in circadian resynchronization in a mouse model of jet lag. J Clin Invest 2010, 120, 2600-2609.
53. Riazanskaia, S.; Blackburn, G.; Harker, M.; Taylor, D.; Thomas, C. L. P., The analytical utility of thermally desorbed polydimethylsilicone membranes for in-vivo sampling of volatile organic compounds in and on human skin. Analyst 2008, 133, 1020-1027.
71
54. Delahunty, T.; Schoendorfer, D., Caffeine demethylation monitoring using a transdermal sweat patch. J Anal Toxicol 1998, 22, 596-600.
55. Kintz, P.; Brenneisen, R.; Bundeli, P.; Mangin, P., Sweat testing for heroin and metabolites in a heroin maintenance program. Clin Chem 1997, 43, 736-739.
56. Huang, C. T.; Chen, M. L.; Huang, L. L.; Mao, I. F., Uric acid and urea in human sweat.
Chinese J Physiol 2002, 45, 109-115.
57. Gallagher, M.; Wysocki, J.; Leyden, J. J.; Spielman, A. I.; Sun, X.; Preti, G., Analyses of volatile organic compounds from human skin. Brit J Dermatol 2008, 159, 780-791.
58. Penn, D. J.; Oberzaucher, E.; Grammer, K.; Fischer, G.; Soini, H. A.; Wiesler, D.; Novotny, M. V.; Dixon, S. J.; Xu, Y.; Brereton, R. G., Individual and gender fingerprints in human body odour. J R Soc Interface 2007, 4, 331-340.
59. Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G., Mass spectrometry sampling under ambient conditions with desorption electrospray ionization. Science 2004, 306, 471-473.
60. Roach, P. J.; Laskin, J.; Laskin, A., Nanospray desorption electrospray ionization: an ambient method for liquid-extraction surface sampling in mass spectrometry. Analyst 2010, 135, 2233-2236.
61. Takats, Z.; Wiseman, J. M.; Cooks, R. G., Ambient mass spectrometry using desorption electrospray ionization (DESI): instrumentation, mechanisms and applications in forensics, chemistry, and biology. J Mass Spectrom 2005, 40, 1261-1275.
62. Talaty, N.; Takats, Z.; Cooks, R. G., Rapid in situ detection of alkaloids in plant tissue under ambient conditions using desorption electrospray ionization. Analyst 2005, 130, 1624-1633.
63. Laskin, J.; Heath, B. S.; Roach, P. J.; Cazares, L.; Semmes, O. J., Tissue imaging using nanospray desorption electrospray ionization mass spectrometry. Anal Chem 2012, 84, 141-148.
64. Ranc, V.; Havlicek, V.; Bednar, P.; Lemr, K., Nano-desorption electrospray and kinetic method in chiral analysis of drugs in whole human blood samples. Eur J Mass Spectrom 2008, 14, 411-417.
65. Wilm, M.; Mann, M., Analytical properties of the nanoelectrospray ion source. Anal Chem 1996, 68, 1-8.
66. Karas, M.; Bahr, U.; Dulcks, T., Nano-electrospray ionization mass spectrometry:
addressing analytical problems beyond routine. Fresen J Anal Chem 2000, 366, 669-676.
67. Hsieh, C. H.; Chang, C. H.; Urban, P. L.; Chen, Y. C., Capillary action-supported
72
contactless atmospheric pressure ionization for the combined sampling and mass spectrometric analysis of biomolecules. Anal Chem 2011, 83, 2866-2869.
68. Kelly, R. T.; Page, J. S.; Luo, Q. Z.; Moore, R. J.; Orton, D. J.; Tang, K. Q.; Smith, R. D., Chemically etched open tubular and monolithic emitters for nanoelectrospray ionization mass spectrometry. Anal Chem 2006, 78, 7796-7801.
69. Harker, M.; Coulson, H.; Fairweather, I.; Taylor, D.; Daykin, C. A., Study of metabolite composition of eccrine sweat from healthy male and female human subjects by H-1 NMR spectroscopy. Metabolomics 2006, 2, 105-112.
70. Wishart, D. S. et al., HMDB: the human metabolome database. Nucleic Acids Res 2007, 35, 521-526.
71. Magnusson, I.; Kihlberg, R.; Alvestrand, A.; Wernerman, J.; Ekman, L.; Wahren, J., Utilization of intravenously administered N-acetyl-L-glutamine in humans.
Metabolism 1989, 38, 82-88.
72. Cody, R. B.; Laramee, J. A.; Durst, H. D., Versatile new ion source for the analysis of materials in open air under ambient conditions. Anal Chem 2005, 77, 2297-2302.
73. Haddad, R.; Sparrapan, R.; Kotiaho, T.; Eberlin, M. N., Easy ambient sonic-spray ionization-membrane interface mass spectrometry for direct analysis of solution constituents. Anal Chem 2008, 80, 898-903.