Quantitative imaging of selenoprotein with multi-isotope imaging mass spectrometry (MIMS)

S. Tang1, C. Guillermier2, 3, M. Wang3, J. Loscalzo1 and C. Lechene2, 3

(1) Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA USA(2) Division of Genetics, Brigham and Women’s Hospital, Boston, MA USA

(3) National Resource for Imaging Mass Spectrometry (NRIMS), Cambridge, MA USA

ACKNOWLEDGEMENTS:C.L. is funded by the NIH grants (5P41EB001974-13, AG034641,R01 AG040019, R21AG034641-01, AG-SS-2215-08, RGP0048, R01 AG040209), HumanFrontier Science Program and the Ellison Medical Foundation. S.T is supported by NIH (R02 HL61795 and 2PO1HL048743-18).

Figure 3: Localization of Se incorporated via Different Pathways in whole cell. HAEC Treated with 150 nM 82Se-Selenite and 10 μM 15N-Thymidine for 4 Days. The acquisition parameters were: field 50 µm, 256x256 pixels, 495 planes, 4 min/plane, 33 hrs total acquisition time. Data are shown for the sum of planes from 1 to 40 and 201 to 241 which correspond to different exploration depth within the whole cell. HAEC cells treated with 76Se-Methylselenocysteine and 77Se-Methionine are not shown here.

CONCLUSION:This preliminary study shows that MIMS is a promising technique for exploring the biosyntesis of selenoprotein in the cells. This specific work allowed determining the best analytical conditions, I.e, sample preparation, validation of Selenium ratio measurement with the Nanosims50l and determination of the analytical conditions for this project. MIMS study will be combined with SEM analysis to recognize the site of protein synthesis with specific organelles within the cell.

HSI 77Se/80Se HSI82Se/80Se

Figure 5. Localization of Se incorporated via Different Pathways in cells embedded fixed lifted sections. (a) HAEC Treated with 600 nM 77Se-Seleno-Methionine for 4 Days. Field 30 µm, 126 planes, 256x256 pixels, 1 min per plane (b) HAEC Treated with 150 nM 82Se-Selenite for 4 Days. Field 40 µm, 579 planes, 256x256 pixels, 2 min per plane. Ratio for 77/80 and 82/80 as a function of the number of incubation days is shown in (c). As for the whole cell, 77Se-Methionine leads to a higher labeling efficiency.

Scheme 1. Metabolic pathway for selenite and SeMet and MeSeCys (1). The biosynthesis of selenoproteins (or seleno-enzymes, circled in red color) requires all selenium sources to convert to a key intermediate metabolic, selenide (H2Se), that is then involved in synthesizing selenocysteine on tRNA molecule, a prerequisite step for selenium to be incorporated into selenoproteins. Abbreviations: selenocysteine (SeCys), selenomethionine (SeMet), methyl-seleno-cysteine (MeSeCys), methylseleninic acid (MSA), methylselenol(MMSe), dimethylselenide (DMSe), trimethylselenonium (TMSe).

Figure 4. Determination of labeling efficiency in various cell compartments. HAEC were exposed to the 3 Selenium isotope-tagged compounds for 4 days. A: 15N/14N and 82Se/80Se HSI image of the cell shown in figure 3 (incubated with 82Se-selenite and 10 µM 15N-thymidine). 15N/14N allows delineating clearly the cell nucleus. The cytoplasm of the cell was compartimentalised into 3 areas cytoplasm 1* (Cyt 1), cytoplasm 2* (Cyt 2), and cytoplasm 3* (Cyt 3) (*defined by an increment of 5 µm away from the nucleus). In each zone, ROIs corresponding to spots (5x5 pixels) of high selenium enrichment were identified. Data for the selenium ratio (multiplied by 10 for clarity) are shown in the 3 graphs for the cytoplasm. Data are for 3 different cells each treated with a specific selenium compounds. Se HSI image of the corresponding cell is shown in b). The labeling efficiency with the various selenium compounds range as follow 600nM 77Se-seleno-methionine> 150nM 82Se-selenite > 300nM 76Se-methyl-seleno-cysteine.

ABSTRACT:Much of the trace element, selenium, is incorporated in mammalian cells as selenocysteine (Sec), the so-called twenty-first amino acid. The selenoproteins synthesized from Sec are limited in number, and, with some key exceptions (the glutathione peroxidases), are largely of unknown function. Furthermore, neither the ideal source of organified selenium for Sec generation nor the compartmental localization of selenoproteins has been investigated in any detail. In response to vascular oxidant stress, human aortic endothelial cells (HAEC) are regulated by endothelial cell antioxidant proteins among which are included the selenoprotein glutathione peroxidases. The synthesis of selenoproteins requires an adequate source and form of selenium for the synthesis of Sec on tRNASec. Selenoprotein synthesis differs with respect to selenium source, as well as cell type and tissue. The source of selenium, its metabolism, and its consequences for specific selenoprotein expression have not yet been explored in any detail, nor has the hierarchical utilization of selenium by source been studied in specific cell types. Multi-isotope imaging mass spectrometry (MIMS) allows high resolution quantitative imaging of stable isotope labels in mammalian cells. We are using MIMS to determine the compartmental localization of stable isotope-tagged selenium compounds as sources for selenoprotein synthesis in HAEC, and to compare the efficiency of labeling from inorganic selenium and various forms of organified selenium to find its ideal source. These sources of selenium were used to enrich the selenium pool in cultured human aortic endothelial cells to study the localization of Se incorporated via different pathways. In a first set of experiments, HAEC were treated with 150nM 82Se-Selenite and 10 μM 15N-Thymidine for 4 Days or treated with 600nM 77Se-Seleno-Methionine and 10 μM 15N-Thymidine for 4 Days. The incorporation of seleno-methionine appears to be more localized around the nucleus when compared to the distribution of selenite. We have done extensive work to precisely identify the selenium isotope peaks by using sodium selenite either alone or mixed with fetal bovine serum. This was tested both on silicon and on gold coated silicon. Selenium measurements on cell sections are now being validated against whole mount cells verifying the absence of selenium loss in sections. The analysis of sections will greatly increase the number of cells analyzed in a given amount of time. MIMS provides a unique and novel way to dissect selenoprotein biosynthesis in cells.

RESULTS:

3 0 0 n M 7 6 S e - M e t h y l - S e l e n o - C y s t e i n e 6 0 0 n M 7 7 S e - S e l e n o - M e t h i o n i n e 1 5 0 n M 8 2 S e - S e l e n i t e

REFERENCES:(1). Suzuki KT, et al (2008) Toxicol Appl Pharmacol 227: 76-83.

a)

b)

HSI 15N/14N

HSI 82Se/80Se

c)

HSI 82Se/80Se ● HSI 12C15N/12C14N

12C14N31P

12C14N 31P HSI 77Se/80Se HSI82Se/80Se

a)

b)

c)

MATERIALS AND METHODS:.Compounds of sodium [82Se]-selenite (996 µg, 98.9% purity), [76Se]-methylselenocysteine (155 µg, 99.9% purity), and [77Se]-seleno-L-methionine (494 µg, 99.8% purity) were provided by N. Suzuki (Chiba University) who used established methods of synthesis.

.Human aortic endothelial cells (HAEC) were grown on Au-coated silicon chips (5 mm x 5 mm) in 6-well plates or directly in 6-well plates. HAEC were incubated with conditioned medium for up to 4 days at 37oC in a 5 % CO2 incubator. There are four conditioned media (1. Buffer; 2. 150 nM 82Se-selenite; 3.6 nM 77Se-seleno-methionine, and 4. 3 nM 76Se-methyl-seleno-cysteine). 10 µM 15N-Thymidine was used to visualize the nucleus.

.At the end of incubation, HAEC on Au-coated silicon chips were rinsed, and freeze vacuum dried, followed by MIMS analysis.

.Alternatively, at the end of incubation, HAEC directly grown in 6-well plates were processed by either one of the following two methods: 1. “Trypsinized and Fixed”: Cells were dissociated by trypsin-EDTA, and then fixed with 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1M cacodylate buffer, and 2. “Fixed and Lifting”: Cells were fixed with the same fixative, and then lifted with propylene oxide. These fixed cells were then embedded, and sectioned (500nm in thickness) for MIMS analysis.

.MIMS using NanoSIMS50 (Cameca) was performed to detect simultaneously mass 12 (12C-), mass 26 (12C14N-), mass 27 (12C15N-) mass 31 (31P-) with mass 76 (76Se-), mass 80 (80Se-) and mass 82 (82Se-) or mass 77 (77Se-), mass 80 (80Se-) and mass 82 (82Se-).

Figure 1. Freeze-drying under vacuum of HAEC cells. Conditions are -80 0C at 10-6 Torr. a) Reflected light dark-field microscopy of HAEC cells grown on silicon chip for two days. b) Schematic of what may happen to the HAEC cells after freeze drying under vacuum. c) CN image of an HAEC cell after freeze drying. Field is 50 µm, 495 planes, 4min/plane.

Whole cell on silicon chip

Freeze Vacuum Drying

nucleus

nucleusa) b)

c)

Cell section on silicon chip

Figure 2. HAEC cell after fixation and sectioning. HAEC, grown in 6-well plates, were processed by two fixation methods: 1. trypsinized and fixed; and 2. fixed and lifted. The fixative was 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1M cacodylate buffer. HAEC were harvested by fixation, embedded and sectioned. (12C14N-) and (31P-) images of HAEC cells, 30 µm field, 178 planes, 2 min/plane for the 2 fixation methods described above. a) trypsinized fixed. b) fixed lifted. Spherical like shapes of HAEC were introduced by trypsin-EDTA treatment. The fixed and lifted preparation method yields to an ideal thin and smooth layer for MIMS analysis.

31P

12C14N 31P

a)

12C14N

b)

SAMPLE PREPARATION:

12C14N

HAEC were incubated with 150 nM 82Se-selenite and 10 μM 15N-thymidine for 4 days.

HSI 77Se/80Se

1-40

201-240

HSI 77Se/80Se HSI82Se/80Se