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Nivedita Natarajan, Padma Ramakrishnan, Vairavan Lakshmanan1, Dasaradhi Palakodeti1, Kannan Rangiah* Metabolomics Facility, Centre for Cellular and Molecular Platforms, 1Institute of Stem Cell Biology and Regenarative Medicine, National Centre for Biological Sciences, GKVK, Bellary Road, Bangalore-560065, India.

* Corresponding author: Metabolomics facility, Centre for Cellular and Molecular Platforms, GKVK, Bellary Road, Bangalore-560065, India. Fax: + 91-80-2363 6662, E-mail:[email protected]

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A Quantitative Metabolomics Peek into Planarian Regeneration

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Abstract Fresh water planarian species Schmidtea mediterranea is an emerging stem cell model because of its capability to regenerate whole animal from a small piece of tissue. It is one of the best model systems to address basic mechanisms essential for regeneration. Here, we are interested to study the role of various amines, thiols and nucleotides in planarian regeneration, stem cell function and growth. We developed mass spectrometry based quantitative methods and validated differential enrichment of 35 amines, 7 thiol metabolites and 4 nucleotides from both intact and regenerating planarians. Among the amines, alanine in sexual and asparagine in asexual are the highest (>1000 ng/mg) in the intact planarians. The level of thiols like cysteine and GSH is 651, 1107 ng/mg in planarians. Among the nucleotides, cGMP is the least (0.03 ng/mg) and AMP is the highest (187 ng/mg) in both the planarian strains. We also noticed increasing levels of amines in both anterior and posterior regenerating planarians. The blastema from day 3 regenerating planarians also showed higher amounts of many amines. Interestingly, thiols (cysteine and GSH) level is well maintained during planarian regeneration. This suggests an inherent and effective mechanism to control induced oxidative stress because of the robust regeneration and stem cell proliferation. Like in the intact planarian, the level of cGMP is very low in the regenerating planarians. Surprisingly, the level of amines and thiols in the head regenerating blastema is ~3 times higher compared to tail regenerating blastema. Thus our results strongly indicate the potential role of amines, thiols and nucleotides in planarian regeneration. Key words: Planaria; metabolites; regeneration; quantification; mass spectrometry; blastema.

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Introduction Regeneration is the ability of an organism to grow a missing piece or a tissue from a tiny body fragment. The process of regeneration is a complex phenomenon, which involves series of multiple cellular events 1. In recent years, planarian flatworms (sub group of Platyhelminthes) emerged as a potential model system to study regeneration due to their amazing regenerative capacity 2. Planarians can regenerate into a whole animal from 1/273rd piece of animal 3. Interestingly the planarians can also grow and de-grow depending on nutritional status

4-5

. The

molecular mechanisms involved in planarian regeneration have long remained a mystery. One of the accepted mechanisms is the presence of planarian pluripotent stem cells called as neoblast and they are distributed throughout the mesenchymal space, from head to tail 6. Regeneration in planaria happens in multiple stages, which involves wound healing, neoblast proliferationdifferentiation, old tissue remodeling and formation of new tissue to regenerate functional planaria 2. Within 24 hour post amputation, the neoblast differentiate to form blastema, which mostly contains progenitor population 7. Blastema is the site where stem cells receive signals to differentiate to either anterior or posterior regenerating tissues 8. Later stages of regeneration involve remodeling of older tissue to readapt to the reduced size by undergoing apoptosis. Several models involving epimorphosis and morphallaxis have been proposed to explain regeneration mechanisms in planaria 3, but it is imperative to identify bio-molecules involved in the regeneration processes. In recent years several molecular tools have been developed to understand planarian regeneration. The availability of a molecular resource database, the sequencing of the genome of S. mediterranea 9, the optimization of genetic interference tools like gene loss function by RNA interference 10-11 and protocols for isolation of neoblast populations contributed significantly to understand the fundamental functioning of the stem cell and regeneration 12. Recently, extensive transcriptome analysis on neoblast populations and regenerating planaria identified several genes that were specifically enriched in neoblasts and regenerating tissue of planaria 13-14. These studies identified several genes that were used as a marker for neoblast (smedwi-1, smed-pcna), axial patterning (Hox genes), dorso-ventral and anterior-posterior patterning genes (Bone morphogenetic proteins, Wnt-pathway proteins), germ cell markers (Nanos-related gene) using ‘proteogenomics’ approach and recent proteomic study from whole planaria 3   

15

14

. By

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16

and neoblast revealed proteins associated with mucous and several neoblast-related

candidate proteins

17-19

. The stable isotope labeling with amino acids in cell culture (SILAC)

based proteomics for planarians stem cells identified nucleolar receptor coactivator-5, which is essential for the maintenance of the pluripotent stem cell population in planarians

20

.

Unfortunately not much is known about the role of metabolites in planarian regeneration and stem cell function. Previous study demonstrated the role of L-proline in regulating the metastability of embryonic stem cells (ESCs)

21

. In a different study, metabolic profiling

identified several metabolites that are specifically enriched in the ESCs. Inhibition of some of the metabolic pathways such as eicosanoid signalling pathway promoted pluripotency of ESCs and increased level of unsaturated fatty acids

22

. However, comprehensive analysis of metabolites

during planarian regeneration and stem cell function has yet to be done. In this present study, we quantified the level of amines, thiols and nucleotides in both intact and regenerating planarians. In this study, we used high-end mass spectrometry with stable isotope dilution (SID) method

23

to quantify metabolites from the intact and regenerating planarian. We have developed quantitative

methods

by

using

ultrahigh

performance

liquid

chromatography-mass

spectrometry/selected reaction monitoring (UHPLC-MS/SRM) with specific internal standards to quantify specific amines, thiols and nucleotides. With these validated methods we have analyzed the level of all these metabolites in intact planarian (in both sexual and asexual strains), in the regenerating asexual planarian (day 0, 1, 3, 5 and 7) and also from the regenerating tissue (blastema from day 3) (Figure 1). Amines were derivatized only with 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AQC) and cysteine thiols were derivatized with AQC after blocking the thiol with iodoacetic acid (IAA) (Figure 2). GSH metabolites were analyzed only by blocking thiol with IAA and nucleotides were analyzed directly without any further derivatization. Our study identified certain amines that were specifically regulated during planarian regeneration suggesting the potential role in the regeneration processes.

Results Synthesis of AQC derivative We have monitored the formation of AQC (286 m/z) by injecting the diluted reaction mixture (10 µg in 1 mL of acetonitrile) into the mass spec through direct infusion. The maximum amount 4   

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of AQC was observed at 20 min and also some amount of AMQ (145 m/z) was left over. By allowing the reaction for another 10 min the peak of AMQ has reacted completely (Figure S1). We always noticed a peak at 171 m/z corresponding to the product ion of AQC, which occurs in the source during the ionization process. We have also confirmed the 171 m/z peak by doing the MS/MS of the parent ion (286 m/z), which showed the same peak as a major product ion. The yield of the reaction is ~80% and the dried powder is stable under vacuum for more than six months.

LC-MS/MS analysis of metabolites All amines showed intense ions corresponding to [M+H] of the relevant AQC derivative. All 35 amines and thiols (cysteine, cystine and cystathionine) and their corresponding deuterated ISTDs showed the reaction with AQC to form the mono-derivative. In case of cysteine the –SH was blocked with IAA prior to the AQC derivatization. For thiols both IAA and AQC derivatization reactions were done in the ammonium bicarbonate buffer instead of borate buffer. The AQC derivatization goes to completion in both buffers at alkali pH 9. Di-derivatization reaction of AQC with the basic amino acids like lysine leads to doubly charged ions (244 Da), lysine-d8 (248 Da) and ornithine (237 Da), ornithine-d7 (240.7 Da). Interestingly the CID based MS/MS analysis of all amines (both mono- and di-derivatives of AQC) showed major single product ion (171 m/z) like the AQC reagent except histidine and histamine. These might be due to the higher ionization efficiency compared to the AQC derived 171-product ion. Melatonin has no primary amine group, and showed no reaction with AQC. Histidine (110 Da), histidine-d3 (113 Da), histamine (138 Da), histamine-d4 (142 Da), melatonin (174 Da) and melatonin-d4 (178 Da) were observed as product ions. The collision energy and the corresponding S-lens voltage and the retention times for all metabolites were showed in the Table 1. In case of GSH thiols, derivatization was done by blocking only with IAA. For thiols, ascorbic acid was used in both stock solution and also in the reaction to prevent the oxidation. In case of nucleotides, the analysis was done directly without any derivatization. All four of nucleotides showed the base moiety as product ions after losing the sugar moiety from the parent compound. The product ions, collision energy and the corresponding S-lens voltage are shown in the Table 1.

Method development 5   

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As such there is no proper matrix to use as a background for planarian extract. We have used the extraction solvents as a matrix for the method development processes. Acid acetone was used as matrix to develop methods for amines and nucleotides. Aqueous acetonitrile was used for cysteine thiols and GSH metabolites. For amines, cysteine thiols and nucleotides were extracted prior to the derivatization, whereas GSH metabolites were extracted after derivatization with IAA. Briefly, both STDs and ISTDs (amines) were spiked in 200 µL of acetone (0.1 % FA, 0.5 mM of ascorbic acid), followed by solvent drying, derivatization reaction with AQC and cleaning it with RP-SPE cartridges. This eluate was further dried and reconstituted in 50 µL of 0.5% acetonitrile and 10 µL was used for the analysis. We have observed the instability of LDOPA, dopamine, epinephrine and norepinephrine under the derivatization condition without ascorbic acid. All amines showed good separation in the reverse phase chromatography and eluted from column from 5 to 26 min. The derivatives were quite stable at least for two days with ascorbic acid under the derivatization condition (data not shown). The AQC reaction with 16 amino acid mix was routinely used to check the efficiency of the derivatization reaction (Figure S2). A typical UHPLC-MS/SRM chromatographic profile of the amines with ISTDs from one of the sample is shown in the figure S3A and S3B.

We have not used specific ISTDs for

asparagine, alanine, arginine, homoserine, octapamine, methionine, isoleucine and tyramine. However, we have used other ISTDs, which elute closely to these metabolites (as mentioned in the experimental section) to construct the STD curve and for quantification from the actual samples. In this derivatization procedure both leucine and isoleucine were baseline separated and leucine-d10 was used to quantify both metabolites. Cysteine, GSH metabolites and ISTD were spiked into 200 µL of 60% acetonitrile with IAA and ascorbic acid to control the oxidation of reduced thiols. For both GSH and cysteine thiols we have used cystathione-d4 as ISTD for quantification. Both IAA and AQC derivatization was done for cysteine metabolites, but only IAA derivatization was done for GSH metabolites. Thiol reaction with IAA is hindered in the borate buffer whereas the AQC derivatization had happened in the bicarbonate buffer. So we have used ammonium bicarbonate buffer for both IAA and AQC derivatization reaction. Under these conditions the oxidation is always minimal (< 5%) for both GSH and cysteine. Since the complete drying of GSH metabolites leads to instability, so analysis was done without drying the solvent starting from extraction to final injection. Cys-gly (8.82 min) and GSH (8.96 min) elutes closely and -glu-cys elutes prior to these two (7.64 min) in the Luna C-18 column. We always 6   

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noticed an extra peak with the similar retention time like GSH in the -glu-cys channel. This might be due to the interference of the product ion of GSH, which is the parent ion of -glu-cys. In case of cysteine thiols both cystine (6.97 min) and cystathionine (6.63 min) elutes prior to cysteine (7.84 min). For nucleotides quantification we have used tyrosine-d7 (2.97 min) as ISTD, which elutes close to AMP and GMP. Both GMP (2.57 min) and AMP (3.08 min), which elute early, showed peak tailing pattern in the reverse phase chromatography (Figure S3C). All metabolites were stable at least for two days at 4 °C (Table S1). The recovery from the SPE cartridge that we have checked for few amines was greater than 80% (Table S2). Calibration curve and limit of quantification Calibration curves for quantifying each amines is linear over a 128-fold, for cysteine thiols 200fold, for GSH thiols 130-fold and for nucleotides 60-fold concentration range with regression correlation coefficients ranging from 0.997 to 1 (Table 1). STD curve constructed for both amines and cysteine thiols were in the ng/ml range. Since the GSH level is very high in the biological system the standard curves were constructed in the μg/mL range concentration. The regression analysis of calibration curves with the mean (± SD) of slope, intercept and the regression constructed on three different days is shown in the table 1. The lower limit of quantification (LOQ), defined as the lowest standard that could be analyzed with an accuracy of 80 to 120% and precision of ± 20% for five replicates on three different days, was 0.31 ng/mL of each amines, 6.25 ng/mL for cysteine thiol metabolites, 0.031µg/mL for GSH thiol metabolites. For nucleotides the LOQ was selected based on the response in the mass spec detection (cAMP0.78, AMP- 0.78, cGMP- 0.1, GMP- 3.91 ng/mL) (Table 1). Assay accuracy, precision and stability Overall, excellent accuracy and precision were obtained for the analysis of all metabolites (Table 1). Inter-day accuracy (n=3) for the LQC (0.625 ng/mL) for all 35 amines ranged from 95106%, for MQC (8 ng/mL) from 94-105% and for HQC (16 ng/mL) from 94-107%. The interday precisions (n=3) were in the range of 2-10% (for LQC), 2-9% (for MQC) and 1-10% (for HQC). For cysteine thiols the concentration of QCs is 20-fold higher compared to amines. Interday accuracy (n=3) for the LQC (12.5 ng/mL) for all three cysteine thiols ranged from 97-107%, for MQC (160 ng/mL) from 100-102% and for HQC (320 ng/mL) from 100-105%. The inter-day precisions (n=3) were in the range of 5-9% (for LQC), 2-6% (for MQC) and 2-7% (for HQC). 7   

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For GSH thiols the concentration of QCs is in the µg/mL range. Inter-day accuracy (n=3) for the LQC (0.062 µg/mL) for all four GSH thiols ranged from 95-108%, for MQC (0.8 µg/mL) from 98-102% and for HQC (1.6 µg/mL) from 96-102%. The inter-day precisions (n=3) were in the range of 2-5% (for LQC), 2-4% (for MQC) and 2-3% (for HQC). For nucleotides inter-day accuracy (n=3) for the LQC (cAMP & AMP- 1.56, cGMP- 0.780, GMP- 7.81 ng/mL) ranged from 105-109%, for MQC (cAMP & AMP- 10, cGMP- 1.5, GMP- 50 ng/mL) ranged from 97107%, for HQC (cAMP & AMP- 20, cGMP- 10, GMP- 100 ng/mL). The inter-day precisions (n=3) were in the range of 0.8 to 3.8% (for LQC), 2-4% (for MQC) and 1.3-4% (for HQC). The details of the method development data are tabulated in the Table 1. The precision and accuracy data for analysis of the LOQ, LQC, MQC, and HQC samples that were re-analyzed after 24 hour standing on the auto sampler gave essentially identical data to that obtained from the original analyses. Analysis of metabolites from sexual and asexual strains of planarian Schmidtea mediterranea Metabolites from planarians in triplicates were processed through the validated UHPLCMS/SRM method. By using validated methods we could categorize amines into three groups purely based on the amounts detected in whole body extract in both sexual and asexual planarians (Figure 3). First category includes 11 amines, which are less than 20 ng/mg in planaria, second category include 11 amines, which are between 20 to 200 ng/mg in planaria and in the third category have 10 amines whose concentration range between 200-1200 ng/mg in planaria. Among the amines, alanine in sexual and asparagine in asexual are the highest (>1000 ng/mg) in the intact planarians. Most of the amines from all the three categories are present at constant levels between sexual and asexual planarians except octapamine, dopamine, methionine and threonine which were >1.5 times higher in sexual planarians. Epinephrine, melatonin and tryptamine were below detection limit in planarians. The cysteine (sexual- 794 ng/mg, asexual510 ng/mg) which is almost 100 times higher than cystine (sexual- 8 ng/mg, asexual- 4.8 ng/mg) and it is 2 times higher than cytathionine (sexual- 392 ng/mg, asexual- 239 ng/mg). Similarly, GSH (sexual- 1212 ng/mg, asexual- 1003 ng/mg) which is higher than cys-gly (sexual- 62 ng/mg, asexual- 78 ng/mg) and -glu-cys (sexual- 226 ng/mg, asexual- 94 ng/mg). GSH is almost 200 times higher than GSSG (sexual- 4.2 ng/mg, asexual- 3.7 ng/mg). Both planarians have the mechanism to maintain the reduced environment where the oxidized metabolites are in very low 8   

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concentration (cystine GMP >cAMP >cGMP which is similar to what has been observed in intact planarians (Figure 5D). Overall, in both posterior and anterior regeneration, each of the nucleotide concentration increases over the 7-day period to attain similar levels as the control intact planarian.

Analysis of metabolites from regenerating blastema Blastema analysis was done from day 3 regenerating planarians. Precise excision of blastema from the regenerating tissue is difficult at day 1 and day 2 post amputation. Whereas in day 3, blastema is clearly visible and easy to excise. Further, It has been shown at day 3 post amputation that neoblast undergo extensive proliferation at the wound region followed by differentiation, which increases the number of blastema cells. Patterning of new and old tissue also starts happening around 2-3 days post amputation 24. Interestingly we could detect almost 32 amines from the blastema of both posterior and anterior regenerating tissues and 14 of the amines are more than 10 ng/mg in either one of the blastemal tissues which is shown in figure 6A. Surprisingly the levels of all these amines were higher in the head regenerating blastema compared to tail regenerating blastema. Asparagine, arginine, aspartic acid, glutamic acid, threonine, lysine, valine and phenylalanine were ~3-fold higher and serine, citrulline, GABA, isoleucine and leucine were ~1.5-fold higher in the head regenerating blastema. We have also quantified the amines from the rest of the tissue after removing the blastema. At least 22 amines 10   

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were more than 10 ng/mg in the rest of the 3 day post amputated tissue after removing the blastema. These amines except leucine, which is 1.5 times higher in anterior regenerating tissue, remained constant level for both regenerating tissues (Figure 6B). Interestingly cysteine, GSH, cystathionine and GSSG were higher in anterior regenerating blastema compared to posterior regenerating blastema (Figure 6C). Both reduced thiols (cysteine and GSH) were 2-times higher in the anterior regenerating blastema, whereas the corresponding tissue other than blastema showed an opposite trend for GSH (Figure 6D). The other thiol metabolites (cystine, cysteine, cystathionine and GSSG) remained constant in the rest of the day 3 regenerating tissue other than blastema. The quantification of all four nucleotides from blastema is not conclusive (data not shown).

Discussions In planarian regeneration the most important question that still remains unanswered is how does a tiny fragment know what to regenerate? Morgan provided the first suggestion of a morphogenetic gradient to account for this process in earthworms

25-28

. In planarians recent

studies have shown existence of anterior-posterior, dorsal-ventral “polarity”, which is maintained throughout the body, will determine the regeneration of the particular cell type or tissue

25-28

.

Again it is important to identify the molecules involved in determining body polarity and its involvement in the regeneration. It has been known that neoblasts are the source of new cells in planaria. Single cell transplantation of neoblast to planaria after radiation treatment, produce cells that differentiate into neuronal, intestinal and other known postmitotic cell types and are distributed throughout the body 29. To understand the cellular basis of regeneration and the role of neoblasts population in differentiation into other cell types become the subject of extensive molecular investigation 30. Recently, several studies have shown the role of metabolites in stem cell function

21-22, 31

. Here, we studied changes in various metabolites, especially amines and

thiols during planarian regeneration, which might provide some clues to understand the role of metabolites in planarian stem cell function and regeneration. It has been known that amines and cysteine thiols are required for many cellular processes notably protein synthesis, pH regulation, synthesis of nucleotides, GSH, nitric oxide, and other signaling molecules, as well as being a potential source of energy

32

. In the regeneration processes maintenance of all the above

mentioned roles of amines are crucial. It has been shown that proline and ornithine are the key 11   

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regulators of ESC metastability and also the redox status of ESCs is regulated during the process of differentiation

21, 22

. It is imperative to quantify amines and thiols in planaria to understand

their role in the regeneration so that it can be applied to individual cell types to understand the differentiation processes. In one of the recent studies it was shown that the human induced pluripotent stem cells share a pluripotent metabolomic signature with ESCs that is distinct from their parental cells

33

. Planarian biology gives us an unique advantage to look at the role of

metabolites during regeneration and in stem cell function. It will also help us to identify the functionally conserved metabolomic signatures that facilitate pluripotency and regeneration across metazoans. One of the best ways to address is through quantitative metabolomics using UHPLC-MS/SRM methods. As of now the quantification of all known metabolites in a single method is still a challenging problem. We have addressed this through quantifying 35 amines, 7 thiols and 4 nucleotides using four different validated methods from intact, regenerating planaria and also from regenerating tissue blastema. Amines are majorly selected in our study because of their potential to regulate key signaling pathways that in turn might potentially regulate regeneration process in planarians. Recent studies also showed anabolism of amino acids, which leads to the synthesis of products that were essential for regulation of stem cell function

34

.

Therefore, we initiated our study by measuring the levels of amino acids during planarian regeneration. To quantify these metabolites we have used SID method 23. This methodology normally involves the use of a stable-isotope-labeled internal standard, which is spiked into a sample at a known concentration. The response ratio between analyte and labeled compound obtained by LCMS/SRM can then be interpolated onto a standard curve to calculate the absolute amount of analyte in the unknown sample. This kind of method is highly selective, sensitive and specific compared to UV, fluorescence and immunoaffinity based methods. Analysis of amines is of great importance and accurate estimation depends on efficiency of extraction, specificity and sensitivity of the method employed. Quantification of amines and thiols directly without derivatization has many difficulties like less binding efficiency to columns, ionization effect in the mass spectrometry and thiol oxidation while sample processing. To avoid all these difficulties we have used pre-column derivatization using AQC derivative. There are many other derivatizing agents like O-phthalaldehyde, phenylisothiocyanate, butylisothiocyanate and dansyl 12   

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chloride used routinely for derivatizing amines 35. We have used AQC for many reasons such as ease of synthesis, simple derivatization which leads to major single product ion and better separation by chromatography. These derivatives can be analyzed directly by mass spec without removing the excess of reagent. This technique is highly sensitive (10-100 fold increase in the sensitivity of amines), accurate and reproducible from small volume of biological sample. This reaction happens mainly with primary amines and the derivatives were stable for longer time. Recently AQC derivatization method was also applied to profile amines from biological fluids based on single product ion (171 Da) formation upon cleavage of derivatives using collision induced dissociation 36. We have developed and validated four different methods to look at amines, thiols (cysteine and GSH metabolites) and nucleotides from planarian extract. So far it was not very clear about amino acid levels and its bio-synthetic pathways in planarians. By using validated methods we could categorize amines into three groups purely based on the amounts detected in planarian whole body extract (Figure 3) and this cannot be extrapolated to other organism or cells. Here we have quantified all the 35 amines (except acetylcholine, glycine, glutamine and selenocysteine) from intact, regenerating planarians and also from blastema at 3 day post amputation. Acetylcholine showed no reaction with AQC, glycine ISTD and glutamine stock stability had a problem and we have not checked selenocysteine in planaria. The amino acid derivatives and neurotransmitters (except serotonin and GABA) are in the range of 0-20 ng/mg category. We observed several amines that were abundant (20 to 1200 ng/mg) in both the strains of planarian Schmidtea mediterranea (Figure 3B & 3C). Among the abundant amines, aspargine, arginine, aspartic acid, glutamic acid, threonine, alanine, lysine, leucine, phenylalanine showed higher levels during planarian regeneration (Figure 4). In addition to the above-mentioned amines serine, citrulline, valine and isoluecine were also present in the blastema of the regenerating tissue (Figure 6A). Interestingly the blastema, which regenerates anterior tissue showed higher amounts of these amino acids compared to posterior regenerating blastema. It seems anterior regeneration in planaria required ~3 fold excess of all these amino acids compared to the tail regeneration. But rest of the corresponding tissue other than blastema showed opposite trend (Figure 6B). We have observed that the concentration of certain amines (asparagine, serine, arginine, aspartic acid, citrulline, glutamic acid, threonine, alanine, aminoadipic acid, lysine, 13   

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tryptophan and histidine) are higher, certain amines (valine, isoleucine, leucine, phenylalanine, hrdroxyproline, octapamine, tyrosine and methionine) are approximately equal and some amines (proline and cysteine) are lower in the planarian regeneration (both posterior and anterior) compared to other part of the tissue (after removing the blastema). This might be due to the presence of blastema, the place where the concentration of certain amines increases or decreases during the regeneration processes. It is interesting to study the role of these amino acids in the regeneration process. Previously, we have shown the presence of all the bio-synthetic pathways that make all neurotransmitters in planaria

37

. Among the neurotransmitters that we have

quantified, both DOPA and dopamine showed greater fold changes during regeneration indicating their role in the planarian regeneration. In this current study using AQC derivatization we have quantified amines which also include neurotransmitters. In the recent report, the growth of mouse ESCs seems to be highly dependant on threonine catabolism 38. In our study we have observed increased levels of threonine, which is one of the abundant amino acids (~300 ng/mg) in planaria, in the regenerating planaria and in the 3 day post amputated blastema. Further, threonine level is almost 3-times higher in anterior regenerating blastema compared to posterior regenerating one suggesting potential role of threonine metabolism in planarian regeneration (Figure 6A). It is also important to look at the levels of threonine in neoblast, which are functionally equivalent to mammalian ESCs. Our study also identified several amines whose levels were significantly down regulated during the initial stages of planarian regeneration (Figure 4C). But interestingly abundant amines in the intact and regenerating planarians also higher in the blastema at day 3 post amputation (Figure 3, 4 & 6A). These results suggest that localized metabolism of these amines in the blastema might be essential for the differentiation of progenitors during planarian regeneration. It is well known that amines are highly essential for many cellular processes either directly in protein synthesis or indirectly in many other amino acid mediated signaling cascades and in neurotransmitter biosynthesis. For instance, mammalian target of rapamycin complex 1(mTORC1), which plays vital role in cell growth regulation and dysregulation is activated by amino acids

39

. It is also well

known that branched chain amino acids (leucine, isoleucine and valine), especially leucine, activate mTORC1. It was reported in planarian that SMED-TOR, SMED-RAPTOR and SMEDLST8 homologs of TOR, RAPTOR and LST8 respectively and members of TORC1, are 14   

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necessary for blastema growth during regeneration

40

. We have quantified all three branched

chain amino acids, which are more than 200 ng/mg in intact planaria. Both valine and isoleucine showed similar pattern in the regenerating planarians whereas leucine is higher in the anterior regeneration (Figure 4C). It will be interesting to look at the role of branched chain amino acids and their effect in the stem cell differentiation in planarians. Our study also detected arginine, ornithine and citrulline suggesting the presence of pathway that is essential for conversion of Larginine to ornithine. All three showed similar levels of change in the regenerating planarians (Figure 4C). Asparagine to aspartic acid and glutamine-glutamic acid-GABA are also present in planarian system. Aromatic amino acids are known to be natural anti-oxidants 41, in addition to their role as a part of protein synthesis. In planarians we detected all four aromatic amino acids (phenylalanine, tyrosine, tryptophan and histidine) in both strains of planaria. Further, phenylalanine showed 2 times higher amount in blastema and regenerating planaria compared to other aromatic amino acids. In planaria, we observed high levels of alanine in intact and regenerating planarians and also in the blastema.

Recent studies have shown alanine

aminotransferase as a biomarker for muscular dystrophy and other muscular diseases. This enzyme catalyzes the transfer of amino group from alanine to α-ketoglutarate and pyruvate in the liver, which is a critical process of the tricarboxylic acid cycle 42. The higher amount of alanine in planaria suggests its role as a major component in the biosynthesis of pyruvate. Similarly, we also observed high levels of lysine in both anterior and posterior regenerating animals. In mammals, lysine the important basic amino acid is mainly involved in the protein synthesis and also the precursor for the biosynthesis of carnitines, which plays an important role in -oxidation 43

. In the head regenerating blastema, lysine which is present in 3-times excess might have some

crucial role to play in the regeneration, which needs to be studied in detail. This data indirectly indicates either the presence or absence of certain pathways in the planarian species S.mediterranea. The formation of DOPA/tyramine indicates the presence of tyrosine hydroxylase/decarboxylase pathway in the planarian model systems. Similarly, the presence of serotonin, GABA and histamine indicates the presence of

enzymes such as tryptophan

hydroxylase, glutamic acid decarboxylase and histidine decarboxylase ezymes essential for their bio-synthesis in planarian system. For instance, glutamate decarboxylase activity has been reported in planarian species Dugesia japonica

44

15   

. The presence of tyramine -hydroxylase and

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tryptophan hydroxylase enzyme essential for DOPA and 5-hydroxytryptophan has also been reported in Dugesia japonica

45, 46

. The higher amount of branched chain amino acids (leucine,

isoleucine and valine) suggest the possibility for the presence of branched-chain alpha-keto acid dehydrogenase complex in planarian system. Similarly, presence of arginine, ornithine and citrulline suggests the presence of active pathway which includes arginase, ornithine decarboxylase and nitric oxide synthase enzymes essential for their conversions.

Previous

studies have also shown the presence of arginine and urea biosynthesis in the land planarian 47. The availability of nutrients, such as amino acids, can influence the formation of specific cell lineages in murine ESCs. It was discovered that mouse ESC are uniquely sensitive to threonine depletion because the conversion of threonine to glycine (for one-carbon metabolism) and to acetyl-CoA (for energy production) is essential for stem cell survival 48. Methionine in eukaryotes is essential for the biosynthesis of S-adenosyl methionine, which is a methyl donor for the methylation of DNA and histones crucial for gene regulation 49. It has been shown that methionine metabolism regulates the maintenance and differentiation of human pluripotent stem cells 50. In planarians we have detected high levels of methionine in planarians and in the regenerating tissue the levels of methionine has increased by 1.2 times. This suggests that methionine metabolism might play an essential role in the regeneration of planaria. We currently need to investigate the levels of S-adenosylmethionine during regeneration to understand the role of methionine to S-adenosylmethionine conversion during planarian regeneration.

Maintenance of reduction-oxidation (redox) pathways has a crucial role in wide

range of biological activities such as immune function, stem cell self-renewal, tumorigenesis and ageing

51

. Planarians, which exhibit robust regeneration capabilities must have developed an

efficient way to overcome the damage induced by free oxygen radicals (Figure 5E). The redox dependent signaling mechanism is a well conserved and presumably ancient pathway primarily used to regulate the levels of free radical, which is based on the ratio of oxidized and reduced forms of crucial cysteine and glutathione residues. Recently, it has been shown that the ratio of GSH/GSSG significantly goes down during embryonic stem cell differentiation. This work suggests that red-ox regulation a key component to regulate oxidative stress occurring during embryonic stem cell differentiation and is important to mediate embryonic stem cell fate

22

. In

planarians, we observed very high levels of cysteine and GSH in intact, regenerating planarians 16   

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and in the 3 day blastema suggesting that planarians constantly maintain unusually high levels of reduced environment to combat the free radicals. In contrast to ESC differentiation, regenerating planarians have extremely high levels of GSH in the blastema and at all time point during regeneration. This also implies that planarians have an efficient system to rapidly convert GSSG and cystine back cysteine and GSH to combat the oxidative stress regenerated during planarian regeneration. Mechanism by which the planarians maintain reduced environment needs to be addressed. One of our hypotheses is that there might be a high turnover of cysteine and -glu-cys mediated by three crucial enzymes -cystathionase, -glutamylcysteine synthetase and GSH synthetase for GSH synthesis (Figure 5E). Similarly, low amounts of GSSG and cystine compared to GSH and cysteine indicate the presence of active metabolic process mediated by the enzymes GSH reductase and cysteine reductase, essential for the conversion of GSSG and cystine back into reduced form in planarians. Further the lower amount of cys-gly indicates there is less bio-degradation of GSH in planarians. Thus, through dissection of metabolic intermediates (cystathionine → cysteine → GSH) suggest a way planarians maintain the reducing environment essential to combat the oxidative stress. Cyclic nucleotides, cAMP and cGMP act as secondary messengers and are known to regulate numerous cell functions such as cell growth and adhesion, energy homeostasis, neuronal signaling and muscle relaxation in eukaryotes

52

. Turnover of cAMP and cGMP crucial for

cellular function is tightly regulated by the interplay of enzymes essential for their biosynthesis and degradation 52. In planarians, the presence and also the role of these secondary messenger during planarian regeneration is not well studied. In this study, we identified both cGMP and cAMP in intact and regenerating tissue of planarians. Interestingly, the level of cGMP is ~ 200fold less compared to cAMP in both strains of planaria. The trend is almost similar even in the regenerating planarians. Indeed our study revealed that the level of cGMP is way below the detection levels suggesting unusually low levels of cGMP in planarians. This could either be due to the low expression of guanylyl cyclase or high expression of the enzymes (cyclic nucleotide phosphodiestrase) essential for their degradation. The other reason could be the restricted expression of guanylyl cyclases to specific tissues and cell types, which need to be further investigated. This result suggests that cAMP has more global function in regulating planarian

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Analyst Accepted Manuscript

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biology compared to cGMP. Similarly, it has been shown that the level of cAMP is much higher in many human cell lines compared to cGMP 53.

EXPERIMENTAL Materials All standards (STDs) like 6-Aminoquinoline (AMQ), N,N′-Disuccinimidyl carbonate (DSC), 4hydroxyproline, histidine, asparagine, taurine, serine, arginine, homoserine, aspartic acid, sarcosine hydrochloride, citrulline, glutamic acid, threonine, alanine, aminoadipic acid, proline, ornithine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine, tryptophan, gamma aminobutyric acid (GABA), histamine, norepinephrine, DOPA, epinephrine, octapamine, dopamine, serotonin, melatonin, tyramine, tryptamine, 18 amino acids mixture, L-Glutathione reduced (GSH), L-Glutathione oxidized (GSSG), -glu-cys, cys-gly, cystine, cysteine, cystathionine, 3’,5’-cyclic adenosine monophosphate (cAMP), adenosine 5’-monophosphate (AMP), 3’,5’-cyclic guanosine monophosphate (cGMP), guanosine 5’-monophosphate (GMP), iodoacetic acid (IAA), ascorbic acid, ammonium bicarbonate, borate salt and ammonium acetate were all obtained from Sigma-Aldrich (Bangalore, India). All internal standards (ISTDs) which are deuterated- L-Serine-2,3,3-d3, L-Glutamic-2,3,3,4,4-d5 acid, L Aspartic-2,3,3-d3 acid, LHistidine-d3 (α-d1; imidazole-2,5-d2) HCl, L-Leucine-d10, L-Lysine-3,3,4,4,5,5,6,6-d8 HCl, NMethyl-d3-glycine-2,2-d2 HCl (Sarcosine hydrochloride), L-Phenylalanine-3,3-d2, L-Proline2,5,5-d3,

L-Threonine-2,3-d2,

L-Tryptophan-2′,4′,5′,6′,7′-d5, L-4-Hydroxyphenyl-d4-alanine-

2,3,3-d3 (tyrosine), L-Valine-d8, L-Ornithine-2,3,3,4,4,5,5-d7 HCl, L-Citrulline-2,3,3,4,4,5,5-d7, DL-2-Amino-1,6-hexanedioic-2,5,5-d3 Acid (Aminoadipic acid), trans-4-Hydroxy-L-proline2,5,5-d3, 2-Aminoethane-d4-sulfonic Acid (Taurine), 4 Aminobutyric-2,2,3,3,4,4-d6 Acid (GABA-d6), Histamine-α,α,β,β-d4, (±)-Norepinephrine-2,5,6,α,β,β-d6, Serotonin-α,α,β,β-d4, (±)Epinephrine-2,5,6,α,β,β-d6, L-Dopa-2,5,6-d3, 2-(3,4-Dihydroxyphenyl)ethyl-1,1,2,2-d4-amineHCl (Dopamine-d4), N-Acetyl-5-methoxytryptamine-α,α,β,β-d4 (melatonin-d4), Tryptamineα,α,β,β-d4 and DL-(2-Amino-2 carboxyethyl)homocysteine-3,3,4,4-d4 (DL-Cystathionine-d4) were obtained from CDN isotopes (Quebec, Canada). The purity of all analytes and deuterated internal standards was ≥98%. High purity MS grade solvents (methanol, acetonitrile and water) were obtained from Merck Millipore (Merck Millipore India Pvt. Ltd., Bangalore). Acetone was obtained from Fisher Scientific and formic acid (FA) was obtained from Sigma-Aldrich 18   

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(Bangalore, India). Solid phase extraction (reverse phase columns) was obtained from Phenomenex, Inc. (Hyderabad, India).

Standard stock preparation All amine STDs and ISTDs were individually weighed and made a stock of 1 mg/mL in 0.1 N HCl and stored at 4˚C. Working stock solution and serial dilutions were also prepared in 0.1 N HCl starting with the concentration 0.5 µg/mL. Derivatizing reagent (1 mg/mL) was prepared in 100% acetonitrile. Thiol STDs (1 mg/mL) were individually weighed and dissolved in water containing 5% ascorbic acid and 3% IAA to control the oxidation. All reduced thiol stocks were prepared fresh for every experiment. Working stock solution and serial dilutions were also prepared in water (containing 5% ascorbic acid and 3% IAA) starting with the concentration 70 µg/mL (GSH metabolites) and 20 µg/mL (Cysteine metabolites). cAMP and cGMP (1mg/mL) were weighed individually and dissolved in 50% methanol, AMP and GMP (1mg/mL) were dissolved in 50% methanol (0.1% FA) and were stored at 4˚C. Both AMP and GMP are prone to degradation under neutral or basic conditions. Working-stock solutions of nucleotide STDs, and serial dilutions were prepared in 50% methanol (with 0.1% FA) starting with a highest concentration of 0.5 µg/mL (for cAMP, cGMP and AMP) and 2.5 µg/mL (for GMP).

Synthesis of AQC reagent Equimolar concentration of AMQ (233 mM-16.8 mg dissolved in 500 µL acetonitrile) and DSC (234 mM-30 mg dissolved in 500 µL acetonitrile) were taken in two separate glass vials. The solution of AMQ was added drop wise to the DSC containing vial and incubated at 60˚C for 30 min. The completion of the reaction was checked by taking samples (1 µL of the reaction mixture in 999 µL of acetonitrile) at different time points (0, 5, 10, 20 and 30 min) and analysed in mass spec through direct infusion. After 30 min the reaction mixture was transferred to eppendorf tube, dried under speed vacuum.

The yield of the reaction was ~80%. It was

calculated by weighing the whitish-yellow colour powder that was obtained after drying. It was then stored in a desiccator under vacuum at room temperature until use.

Derivatization protocol for metabolites

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Amines:

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Both STDs and ISTDs were spiked in 80 µL borate buffer (200 mM, pH 8.8)

containing 1 mM ascorbic acid. To the above mixture 10 µl of AQC (from 1 mg/mL stock) was added and incubated at 55˚C for 10 min. Reaction was stopped by reducing the pH to ~3.5 by the addition 500 µL of acidic water (0.1% FA). The amine derivatives were purified by Reverse Phase-Solid Phase Extraction (RP-SPE) cartridges (Phenomenex, Inc., Hyderabad, India). Briefly RP-SPE cartridges were activated by 1 mL methanol and equilibrated with water (0.1% FA). Samples were loaded on to SPE cartridge and allowed to bind with the matrix. It was then washed twice with 1 mL of acidic water (0.1% FA) and final elution with 1 mL of acetonitrile:methanol (80:20) containing 1% FA. It was again dried and reconstituted with 50 µL of 0.5% acetonitrile and from which 10 µL was injected for the UHPLC-MS/SRM analysis. Cysteine metabolites: Two step derivatization procedures were used to quantify cysteine thiols. The first step was to protect thiol oxidation by IAA and the second step was to derivatize the amine group by AQC. Both STDs and ISTDs were spiked in 100 µL ammonium bicarbonate buffer (200 mM, pH 9) containing 70 mM IAA and 25 mM ascorbic acid. Further, the pH of the solution was adjusted to 9 by addition of 1 µL ammonium hydroxide. The reaction mixture was placed in dark at room temperature for 25 min. The second step of the reaction was initiated by the addition of 20 µl of AQC (from 1 mg/mL stock) to the above solution and incubated at 55˚C for 10 min. Further stopping the reaction, SPE purification and analysis were done by the similar procedure as mentioned above. GSH metabolites: In case of GSH metabolites we have used one-step derivatization with IAA to protect thiols. Both standards and internal standards were spiked to 100 µL of ammonium bicarbonate buffer (100 mM, pH 9) containing 20 mM of ascorbic acid 100 mM of IAA. After addition of ascorbic acid the pH of the solution was adjusted to 9 by the addition of 1.5 µL ammonium hydroxide. The reaction mixture was kept in dark at room temperature for 30 min and reaction was stopped by the addition of 1 µL of FA and followed by the addition of 150 µL of 60% acetonitrile. From this 10 µL of the supernatant was directly injected after centrifugation (5 min, 10000 rpm) into UHPLC-MS/SRM analysis.

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Nucleotides: All four nucleotides were directly analyzed by mass spectrometry without any derivatization. In this case both STDs and ISTDs were spiked into 200 µL of acidified acetone (acetone with 0.1% FA and 0.5 mM ascorbic acid). These were dried under vacuum and reconstituted in 50 µL of 0.5% methanol (with 0.05% FA). 10 µL of each were injected for UHPLC-MS/SRM analysis.

LC-MS The mass spectrometer used for the metabolite analysis is a Vantage TSQ triple stage quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with heated electrospray ionization (HESI). The mass spectrometer is coupled with an Agilent 1290 infinity UHPLC system (Agilent Technologies India Pvt. Ltd., India). This UHPLC system is provided with column oven (set at 40º C), auto-sampler and a thermo-controller (set at 4°C). It uses a flow through injection mode and is equipped with a needle wash system (with acetonitrile, 0.1% formic acid) before injection to ensure zero percent carry over problems. Four different methods were setup in both UHPLC and in the MS system for the analysis of metabolites of all four categories. Same solvent system was used in all four methods. The mobile phase- Solvent A was water (10 mM Ammonium acetate) containing 0.1% formic acid and Solvent B was acetonitrile containing 0.1% formic acid. Amines: In the LC system we have used a C-18 column (2.1 × 100 mm, 1.8 μm, Agilent, Inc). The gradient was optimized to get maximum separation (2% B at 0 min, 2% B at 3 min, 20% B at 20 min, 35% B at 25 min, 80% B at 25-27 min, 2% B at 27-35 min) at 200 L/min flow and we were able to achieve baseline separation between the isomers leucine and isoleucine. Operating conditions were as follows: spray voltage-3700 V; ion transfer capillary temperature270 °C; source temperature- 30 °C; sheath gas-25, auxiliary gas-10 (arbitrary units); collision gas-argon; S-lens voltage and collision energy which was optimized for individual metabolites were incorporated in the method; scan time of 50 millisec/transition and ion polarity positive. Cysteine metabolites: The same C-18 column as mentioned above was used for the separation of cysteine metabolites. The gradient was optimized to get maximum separation at (2% B at 0 min, 2% B at 3 min, 20% B at 7 min, 35% B at 15 min, 80% B at 18-20 min, 2% B at 20.1-25 21   

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min) at a flow rate of 200 L/min. Operating conditions were as follows: spray voltage-3500V; ion transfer capillary temperature-270 °C; source temperature- 80 °C; sheath gas-20, auxiliary gas-10 (arbitrary units); collision gas-argon; S-lens voltage and collision energy which was optimized for individual metabolites were incorporated in the method; scan time of 50 millisec/transition and ion polarity positive. GSH metabolites: In the LC system we have used a Luna C-18(2) column (4.6 × 150 mm, 5 μm, Phenomenex, Inc). The gradient was optimized to get maximum separation at (0% B at 0 min, 0% B at 3 min, 80% B at 8 min, 80% B at 8-9 min, 0% B at 9.1-20 min) at a flow rate of 300 L/min. Operating conditions were same as cysteine metabolites. Nucleotides: In the LC system we have used a Kinetex PFP column (2.1 × 150 mm, 1.7 μm, Phenomenex Inc.). The gradient was optimized to get maximum separation at (0% B at 0 min, 0% B at 2 min, 30% B at 13 min, 80% B at 13.1 min, 80% B at 15 min, 0% B at 15.1 min, 0% B at 22 min) at a flow rate of 150 L/min. MS operating conditions were as follows: spray voltage3500V; ion transfer capillary temperature- 270 °C; source temperature- 300 °C; sheath gas- 20, auxiliary gas-10 (arbitrary units); collision gas- argon; S-lens voltage and collision energy optimized for individual metabolites were incorporated in the method; scan time of 50 millisec/transition and ion polarity positive. To obtain details of tandem mass spectrometry (MS/MS) scans, syringe pump was used with flow of 5 µL/min along with T valve and a solvent flow rate of 200 µL/min. For each metabolite the precursor ion was monitored and collision-induced dissociation (CID) was used to generate product ions. The product ions were obtained by scanning the quadrupole-3 from m/z 50 to 500 with a cycle time of 1 sec. This was followed by optimization of S-lens voltage and collision energy for each of the intense product ions as shown in the table 1.

Calibration curves Amines: The range for the calibration curves was 0.14 ng/mL to 20 ng/mL. All ISTDs were at the concentration of 20 ng/mL. To extract amines from the actual sample we have used 200 µL acid acetone (0.1% FA) containing 0.5 mM ascorbic acid. Similarly, to construct the standard 22   

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curves, both STDs and ISTDs were also spiked in the same solvent and dried in speed vacuum. It was then reconstituted in 80 µL borate buffer (200 mM, pH 8.8) containing 1 mM ascorbic acid and followed the same derivatization protocol as mentioned above. For some analytes we do not have the corresponding ISTDs. In these cases we have used other ISTDs which elutes closely to those analytes. For asparagine, arginine, homoserine- serine-d3, for alanine- aminoadipic acid-d3, for octapamine- glutamic acid-d5, for methionine- valine-d8, for tyramine- ornithine-d7 were used as ISTDs. Daily eight point calibration samples (0.15, 0.31, 0.62, 1.25, 2.50, 5, 10 and 20 ng/mL) were prepared and analyzed together with five replicates of low limit of quantification (LOQ- 0.3 ng/mL) and QC samples (LQC- 0.62 ng/mL, MQC- 8 ng/mL and HQC- 16 ng/mL respectively). Cysteine metabolites: To control the oxidation of thiols, both STDs and ISTDs were spiked in 200 µL of 60% acetonitrile (containing 0.1% FA and 10 mM of ascorbic acid) and dried under speed vacuum. It was then reconstituted in 100 µL ammonium bicarbonate buffer (200 mM, pH 9) containing 70 mM IAA and 25 mM ascorbic acid and followed the same procedure as mentioned above in the derivatization step. The range for the calibration curves was 3.1 ng/mL to 400 ng/mL. We have used cystathionine-d4 as ISTD at the concentration of 400 ng/mL to construct the STD curve and to quantify all cysteine metabolites. Daily eight point calibration samples (3.1, 6.2, 12.4, 25, 50, 100, 200 and 400 ng/mL) were prepared and analyzed together with five replicates of low limit of quantification (LOQ- 6.2 ng/mL) and QC samples (LQC- 12.4 ng/mL, MQC- 160 ng/mL and HQC- 320 ng/mL respectively). GSH Metabolites: The range for the calibration curves was 0.015 µg/mL to 2 µg/mL. Here also we have used cystathionine-d4 as ISTD at the concentration of 1.2 µg/mL to construct the STD curve and to quantify all GSH metabolites. These curves were prepared by spiking all STDs and ISTD as per the protocol mentioned in the derivatization step. Daily eight point calibration samples (0.015, 0.031, 0.062, 0.125, 0.25, 0.25, 0.5 and 1 and 2 µg/mL) were prepared and analyzed together with five replicates of low limit of quantification (LOQ-0.031 µg/mL) and QC samples (LQC- 0.062 µg/mL, MQC- 0.8 µg/mL and HQC- 1.6 µg/mL respectively) and LOD was 0.015 µg/mL.

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Nucleotides: The range for the calibration curves was: 0.05ng/mL to 12.50ng/mL (cGMP), 0.39 ng/mL to 25 ng/mL (cAMP and AMP), and 1.95 ng/mL to 125 ng/mL (GMP). Tyrosine-D7 was used as an ISTD at a concentration of 25 ng/mL, to construct the STD curve and to quantify all nucleotide metabolites. These curves were prepared by spiking all STDs and ISTD as per the protocol as mentioned in the above protocol section. Daily, nine-point calibration samples (0.04, 0.09, 0.19, 0.39, 0.78, 1.56, 3.12, 6.25 and 12.5 ng/mL for cGMP), and seven-point calibration samples (0.39, 0.78, 1.56, 3.12, 6.25, 12.5 and 25 ng/mL for cAMP and AMP), and (1.95, 3.91, 7.81, 15.62, 31.25, 62.5 and 125 ng/mL for GMP), were prepared and analyzed together with five replicates of low limit of quantification (LOQ: 0.09 ng/mL (cGMP), 0.78 ng/mL (cAMP and AMP) and 3.9 ng/mL (GMP)) and QC samples: LQC- 0.78 ng/mL (cGMP), 1.56 ng/mL (cAMP and AMP) and 7.8 ng/mL (GMP), MQC- 1.56 ng/mL (cGMP), 10 ng/mL (cAMP and AMP) and 50 ng/mL (GMP), and HQC- 10 ng/mL (cGMP), 20 ng/mL (cAMP and AMP) and 100 ng/mL (GMP).

Method validation Rigorous bio-analytical validation was conducted using criteria established by the US FDA. Calibration linearity was studied using internal standard spiked calibration solution at eight concentrations for amines and thiol metabolites, for nucleotides nine different concentrations were used. Integrated peak area of the selected SRM transitions were used to build the standard curves. For AQC derivatives the common product ion (m/z 171) was used to build the STD curve. Curves were fitted by an equal weighted regression analysis using the quantification software (X-calibur, version 2.22). Precision and accuracy were evaluated using four concentration points (LOQ, LQC, MQC and HQC). Accuracy of 85–115% and precision of ±15% were considered acceptable for LQC, MQC and HQC samples. Accuracy of 80–120% and precision of ±15% were considered acceptable for the LOQ as recommended. Five replicates of each point were analyzed to determine the intra- and inter-day accuracy and precision. This process was repeated over 3 days in order to determine the inter-day accuracy and precision using the freshly prepared calibration curves. Accuracy was determined by the recovery of QC, and the precision was expressed as the coefficient of variation (CV) of the determination of QCs. Inter-day accuracy and precision were calculated similarly for the 15 replicates of each

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Analyst Accepted Manuscript

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DOI: 10.1039/C4AN02037E

concentration point pooled from the three validation runs. Carryover, stability in the autosampler vial was also checked.

Planarian extract preparation Two strains of fresh water planarians (sexual and asexual) from the species S. mediterranea were maintained in the laboratory at 20 °C in double distilled water which contains all necessary salts (1 M sodium chloride, 0.2 M calcium chloride, 0.2 M magnesium sulphate, 0.2 M magnesium chloride, 0.2 M potassium chloride and 0.02 M sodium bicarbonate). Planarians were fed with beef liver homogenate and allowed to grow ~1 to 2 cm size. Planarians were starved for a week prior to the experiment. Three planarians in triplicates from both sexual and asexual strains were washed in distilled water thrice and weighed using an accurate balance (ME Micro Balance ME36S, Sartorius) after removing the water. Each sample of three planarians was crushed using plastic pestle in 200 µL acid acetone (0.1% FA) containing 0.5 mM ascorbic acid and followed by sonication for 5 min and centrifugation for 10 min at 14000 rpm, 4°C. From the supernatant 50 µL was used for amines and the other 50 µL for nucleotide analysis. To this ISTDs were added and followed the same protocol as mentioned previously. To calculate the GSH and cysteine metabolites another set of planarians under the same conditions in triplicates of three planarians each were crushed in 100 µL of water (0.1% FA) with 20 µL ascorbic acid (from 100 mM stock) and followed by sonication and centrifugation (14000 rpm, 4°C for 10 min). From the supernatant 50 µL was used for GSH metabolites and another 50 µL for cysteine metabolites. The final quantification was done as per the protocol used for standards.

Quantification of amines, thiols and nucleotides in the regenerating asexual planarians and from day 3 blastema Amines, thiols and nucleotides analysis was performed at different time points by cutting the planarians into two pieces (head: above the pharynx and tail: which includes the pharynx) and allowed to regenerate for 7 days. Three intact planarians in triplicate were taken as a control. After cutting the planarians, both the head and tail regions were allowed to regenerate separately in the same buffer system. Briefly, the sample preparation was done in the same way as mentioned above by taking three planarians each in triplicate from the head and tail regions of the regenerating planarians. The UHPLC-MS/SRM analysis for both the head and tail regions 25   

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Analyst

Analyst

   

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DOI: 10.1039/C4AN02037E

was done separately at all time points (2 hour, days 1, 3, 5 and 7). The samples were weighed before extraction. After collecting the samples, it was processed till derivatization followed by cleaning and then stored at -4 °C. The final UHPLC-MS/SRM analysis of amines and nucleotides was done together for all the samples at the end of the experiment. But for both cysteine and GSH metabolites the analysis was done within 48 hour after collecting the samples. Intact planarian controls were also taken to check the level of thiols. Amines, thiols and nucleotides analysis from blastema was performed by cutting the animals into two pieces (head: above the pharynx and tail: which includes the pharynx) and allowed to regenerate for 3 days. The blastema was dissected from the rest of the body tissue directly by cutting the tissue under microscope. The blastema and rest of the body tissue were collected from nine planarians in triplicate for the analysis of amines, thiols and nucleotides separately. The tissues were weighed prior to the metabolite extraction and analysis was done in the same way as above. Since drying leads to inaccuracies in the thiol estimation, the thiols were extracted directly into 0.1% FA without weighing. The weight obtained for blastema in the amine quantification was used for thiol since we have used similar size and number from day 3 regenerating planarians.

Clustering analysis To check the fold changes in the level of amines at different regeneration time points (day 0, 1, 3, 5, 7) compared to intact planarian control, we have done single linkage hierarchical clustering using cluster 3.0. To visualize the heat map we used Java TreeView. Statistical significance (pvalue