Blood phenylalanine reduction reverses gene expression changes observed in a mouse model of phenylketonuria

Phenylketonuria (PKU) is a genetic deficiency of phenylalanine hydroxylase (PAH) in liver resulting in blood phenylalanine (Phe) elevation and neurotoxicity. A pegylated phenylalanine ammonia lyase (PEG-PAL) metabolizing Phe into cinnamic acid was recently approved as treatment for PKU patients. A potentially one-time rAAV-based delivery of PAH gene into liver to convert Phe into tyrosine (Tyr), a normal way of Phe metabolism, has now also entered the clinic. To understand differences between these two Phe lowering strategies, we evaluated PAH and PAL expression in livers of PAHenu2 mice on brain and liver functions. Both lowered brain Phe and increased neurotransmitter levels and corrected animal behavior. However, PAL delivery required dose optimization, did not elevate brain Tyr levels and resulted in an immune response. The effect of hyperphenylalanemia on liver functions in PKU mice was assessed by transcriptome and proteomic analyses. We observed an elevation in Cyp4a10/14 proteins involved in lipid metabolism and upregulation of genes involved in cholesterol biosynthesis. Majority of the gene expression changes were corrected by PAH and PAL delivery though the role of these changes in PKU pathology is currently unclear. Taken together, here we show that blood Phe lowering strategy using PAH or PAL corrects both brain pathology as well as previously unknown lipid metabolism associated pathway changes in liver.

Delivery of PAH and PAL genes to livers normalizes amino acid imbalance, brain neurotransmitter levels and corrects behavioral defect in PAH enu2 mice. The PAH and PAL treatment groups dosed at 3e11 were used for biochemical brain analyses (n = 5 per group). High brain Phe in PKU naïve mice (102.9 ± 7.5 µM) was reduced to normal levels after PAH (14.18 ± 1.82 µM) and PAL treatment (9.62 ± 1.07 µM) while the increase in brain Tyr from 6.74 ± 0.46 µM to 12.1 ± 1.71 µM was observed only in PAH treated mice (Fig. 2). No significant differences were observed in brain Tyr in the PAH enu2 naïve and HET mice despite 30-40% higher blood Tyr levels in HET mice. Brain tryptophan (Trp) levels also increased from 2.16 ± 0.15 to 2.88 ± 0.29 µM with PAH treatment and to 3.06 ± 0.25 µM with PAL treatment (Fig. 2). Analysis of brain neurotransmitter levels showed a marked increase in L-DOPA, dopamine and norepinephrine levels in both PAH and PAL treated mice (Fig. 3B-D). Similarly, a significant increase of 5-hydroxytryptophan, serotonin and serotonin metabolite 5-hydroxyindolactetic acid (5-HIAA) levels were observed after expression of either PAH or PAL in comparison to naïve PKU mice ( Fig. 3F-H).
Change in neurotransmitter levels has been associated with neurobehavioral phenotypes in PKU patients and mice. We measured the ability of PAH enu2 and HET mice to build nests, a social behavior commonly seen in normal mice. Mice were scored on a scale of 1-5 based on their ability to form a nest. Baseline pretreatment nesting behavior was significantly lower in most naive PAH enu2 mice compared to the normal HET mice (Fig. 4A). After 34 days of treatment, a significant improvement was observed in nest building ability of PAH and PAL treated mice while untreated mice still performed poorly (Fig. 4B). The pre-and post-treatment scores for each individual mouse are shown in Fig. 4C.
Transcriptome analysis of livers of PAH enu2 mice. To obtain a more in-depth look at the disease mediated changes and the differences in treatment strategies, we performed high-throughput transcriptome sequencing (RNA-seq) analyses in the livers of PAH enu2 mice. Comparison of gene expression in the liver of naïve PAH enu2 mice to HET mice revealed 1062 differentially expressed genes (p < 0.05 and fold change ≥ 1.5) of which 658 genes were upregulated while 404 genes were downregulated. Treatment with PAH reduced the differentially expressed genes (DEG) to 297 (p < 0.05 and fold change ≥ 1.5) with 171 upregulated and 126 downregulated while PAL treated mice showed 472 differentially expressed genes (p < 0.05 and fold change ≥ 1.5) of which 324 were upregulated and 148 downregulated (Fig. 5A). Of the 1062 DEG in naïve PAH enu2 mice, expression of 887 genes was corrected by PAH treatment and 921 by PAL treatment suggesting that both treatments effectively www.nature.com/scientificreports/ corrected majority of the gene expression changes (Fig. 5B). On comparing the gene expression changes between the two treatment groups, we observed a significant upregulation of genes immunoglobin encoding genes in PAL treated mice that was not observed in PAH treated mice ( Supplementary Fig. S2E). Mononuclear inflammatory cells were considered the source of these upregulated genes. Next, we performed Ingenuity Pathway Analysis (IPA) to study specific pathways impacted by the differentially expressed genes (DEG). Pathways associated with cholesterol biosynthesis were found to be the most affected in untreated PAH enu2 mice (Fig. 5C). Modest but consistent upregulation was observed for the majority of genes at various steps of cholesterol biosynthesis pathway in naïve PAH enu2 mice (Fig. 5D). This included small upregulation of sterol regulatory element binding transcription factor 2 (SREBF2), a master-regulator of the cholesterol pathway while sterol regulatory element binding transcription factor 1 (SREBF1), more involved in regulation of fatty acid synthesis was slightly downregulated (Fig. 5D, Supplementary Fig. S3A). Treatment with PAH and PAL lead to the correction of most genes involved in cholesterol biosynthesis to normal levels suggesting that the changes in cholesterol biosynthesis pathway were linked to the disease (Fig. 5D, Supplementary  Fig. S3A). RT-PCR validation of key genes of cholesterol biosynthesis corroborated the findings from RNA-seq ( Supplementary Fig. S3A).
The top 6 upregulated and downregulated genes in PAH enu2 mice as compared to normal HET mice are shown in Fig. 5E. One striking observation was a massive upregulation (76-fold) of Cyp4a14 in naive PAH enu2 mice, a gene encoding a cytochrome P450 monooxygenase (Fig. 5E). The overexpression of this gene and Cyp4a10, another P450 mono-oxygenase were confirmed by RT-PCR ( Supplementary Fig. S3B). No upregulation of Cyp4a14 and Cyp4a10 were detected in the PAH and PAL treatment cohorts (Fig. 5F, Supplementary Fig. S3B). To observe potential upstream and downstream impact to Cyp4a14 upregulation, we analyzed the expression of Peroxisome proliferator-activated Receptor α (PPARα) and Fatty acid translocase (FAT)/CD36. Both genes were upregulated in naïve PAH enu2 mice and subsequently downregulated with PAH and PAL treatments (Fig. 5F, Supplementary Fig. S3B).
Proteomic analysis of livers of PAH enu2 mice. To confirm that the changes in the liver gene expression profiles translated to changes at the protein level, we performed another study to analyze the proteome of the livers of PAH enu2 mice. Proteomic analysis was performed on PAH enu2 mice, PAH enu2 mice treated with either PAH or PAL at the dose of 1e11 and HET mice ( Supplementary Fig. S1A). A total of 355 proteins (202 upregulated and 202 downregulated) were differentially expressed in the livers of PAH enu2 mice as compared to HET mice while PAH and PAL treated PKU mice showed fewer deregulated proteins (Fig. 6A). Similar to the RNA-seq results, Cyp4a10 protein was among the top upregulated proteins in PAH enu2 mice along with Cyp17a1 which is mainly involved in steroid biosynthesis (Fig. 6C). Cyp4a14 was also ninefold upregulated (p value = 0.051) in PAH enu2 mice but was excluded from our analysis as it was slightly above our cutoff criteria of statistical significance (p ≤ 0.05). Other top upregulated proteins included Mtnd2, a mitochondrial protein coding for NADH dehydrogenase and Acmsd, an enzyme involved in NAD + regulation and maintaining mitochondrial homeostasis. The IPA analysis of deregulated proteins in PAH enu2 mice relative to HET mice identified LPS/IL-1 mediated inhibi- Brain Tyr levels are restored to normal in PAH PAH enu2 mice but not in PAL PAH enu2 mice. (C) Brain Trp is increased in both PAH and PAL treated PAH enu2 mice. N = 5 per group were used for analysis. Control mice and mice treated with 1e11 vg/mouse of PAH or PAL vector were terminated on day 41 and perfused with PBS. Naïve untreated PAH enu2 mice, PAH or PAL, treated PAH enu2 mice, HET untreated HET mice. One-way ANOVA Tukey's multiple comparison, **p < 0.01 and ****p < 0.0001. www.nature.com/scientificreports/ tion of RXR function, (downstream function of this pathway is lipid and xenobiotic metabolism), xenobiotic metabolism PXR signaling and glutathione mediated detoxification among the top affected pathways (Fig. 6B). The expression of top three proteins in each of these affected pathways is shown in Supplementary Fig. S4A-C). All these proteins were upregulated in PAH enu2 mice and either normalized or trending towards normalization post PAH and PAL treatment. We also observed modest changes in various proteins involved in cholesterol biosynthesis ( Supplementary Fig. S4D) consistent with the gene expression results above. Next, we performed a network analysis to understand the interaction between the differentially expressed proteins in our data set. This analysis assumes that highly connected networks are biologically significant. The network with highest score was lipid metabolism suggesting that the greatest number of differentially expressed proteins in the liver of PAH enu2

Discussion
Reduction of blood Phe levels has been the main treatment goal in PKU patients to minimize toxic Phe effect on brain 16,19,20 . The current standard of care to achieve this is the consumption of low Phe diet which is initiated soon after birth to prevent severe brain damage. However, compliance with strict diet steadily decreases with age and among teens and adults, the majority of the patients have higher than recommended Phe levels 8,9 . The only other approved therapy for severe PKU patients currently is PEGylated PAL that is a non-mammalian enzyme converting Phe into trans-cinnamic acid [13][14][15] . Though efficacious, the therapy requires daily s.c. administrations, long titration period and the patients often develop immune responses both the PEG and PAL 21 . Multiple other therapies are currently in development including rAAV-based PAH gene replacement to provide a normal Phe metabolizing pathway in a more sustained and stable manner 16 .
To better understand potential differences between the two strategies for Phe metabolism and to study the effects of elevated Phe on liver and brain, we delivered PAL and PAH genes into livers of PAH enu2 mice, a model of human PKU. Our data demonstrated that viral mediated gene transfer of either gene resulted in reduced blood  www.nature.com/scientificreports/ Phe levels, though the use of PAL over-corrected when high doses were administered. Hence the use of PAL will require careful optimization to obtain normal blood Phe levels as is currently performed for PEGylated PAL in the clinic 15 . This is not surprising as PAL is not subject to allosteric regulation by blood Phe levels. In contrast, multiple studies have demonstrated fine-tuning of PAH activity by Phe binding to N-terminal regulatory domain of PAH 22,23 . At lower Phe levels, the enzyme is maintained as a less active dimer form while with increasing Phe levels the enzyme is driven into a more active tetramer form 22,24 . As expected, only PAH treatment increased the Tyr levels in the blood. The mechanism by which high Phe causes neurotoxicity in PKU is unclear. Potential explanations include reduced amino acid transport into brain and subsequent reduction in brain neurotransmitter levels 4,25 . High blood Phe competes with the transport of large neutral amino acids (including Tyr and Trp) into brain causing overall lowered levels of these amino acids in brain 4,25 . We observed this more for Trp than for Tyr and this may have been due to the use of normal rodent diet. High Phe levels have also been reported to inhibit protein production and enzyme activities, particularly the enzymes involved in neurotransmitter synthesis 25 . Our brain analyses of animals treated with PAL and PAH vectors showed that both treatments normalized brain Phe levels due to reduction of Phe in the blood. Both treatments also corrected brain Trp levels indicating a better large amino acid transport to brain. The LAT1 transporter has been reported to have high affinity to Phe resulting in lowered amino acid transport of large neutral amino acids in the presence of high Phe 26 . Only PAH provided elevated Tyr in the brain as expected due to higher Tyr measured in blood. Despite animals being fed the normal diet, the PAL treated mice exhibited low blood Tyr levels which corresponded to lower-than-normal Tyr in the brain (Figs. 1D,E, 2B). This was expected to result in lower dopamine levels since Tyr is a substrate for dopamine synthesis. However, both PAL and PAH treatments normalized brain dopamine levels suggesting that Tyr deficiency is not the main cause for reduced brain dopamine levels in PKU mice. Other products in the dopamine pathway such as L-DOPA and norepinephrine were also consistently increased by both treatments (Fig. 3B,D). While the low Tyr levels in the PAL treatment group did not seem to be limiting for dopamine synthesis the higher Tyr in PAH treatment group may be beneficial during suboptimal Phe reduction. Interestingly, serotonin and its intermediates and breakdown products had a much higher magnitude of correction with PAH and PAL than those in the dopamine pathway. Furthermore, there was a trend of PAL treatment providing higher levels www.nature.com/scientificreports/ of neurotransmitters which may have been due to slightly lower blood and brain Phe levels (Fig. 3). This suggests that neurotransmitter synthetizing enzymes are highly sensitive to Phe levels in the brain. Unlike the PAH delivery, the delivery of PAL vector resulted in detectable immune response in liver. An infiltration of inflammatory cells, mostly consisting of B-cells, was observed in all PAL treated animals using histopathology. The nature of these cells as B-cells was also supported by RNA seq analysis of liver that showed upregulation of immunoglobulin genes. The immune response was not unexpected since PAL is a bacterial protein. The material used in the clinic has been PEGylated to reduce the immune response. Despite this, high and sustained levels of antibodies both to PAL and PEG were observed in PKU patients and hence, PKU patients using PEG-PAL (Palynziq) are required to carry EpiPen as a precautionary measure 21,27 .
Though neurotoxicity is the hallmark of PKU pathology, we attempted to understand the effect of hyperphenylalanemia in the liver, the major site for PAH expression. Both the transcriptome and proteomics data revealed changes in large number of genes (Figs. 5A, 6A). Most of these changes were reversed post Phe normalization by PAH and PAL treatments suggesting that these changes were caused by elevated Phe levels and hence were disease specific changes (Figs. 5A,B, 6A). Both analyses demonstrated the cholesterol synthesis pathway was upregulated in PKU with elevated expression levels of many enzymes involved in this complex multistep pathway (Fig. 5C,D, Supplementary Fig. S4D). These included SREB2, the master-regulator of the pathway while the SREB1, controlling the fatty acid synthesis was reduced. This upregulation could be in response to the low total cholesterol levels in sera of PAH enu2 mice and PAH-KO mice observed by us and others 28,29 . In PKU patients, serum cholesterol, HDL, LDL are lower than in healthy controls 30 . Similarly, long-chain unsaturated fatty acids levels such as docosahexaenoic acid (DHA) and arachidonic acid (AA) have been reported to be lower in PKU patients 30 . The lowered cholesterol has been proposed to be caused by the inhibition of Phe or its metabolites on HMG-CoA reductase and mevalonate 5-pyrophosphate decarboxylase 28,31 .
The highest degree of over expression in the PKU naïve mice livers was from CYP4A family (Cyp4a10, Cyp4a14 and Cyp17a1) of heme-containing monooxygenases that are largely involved in oxidation of lipids (Figs. 5E, 6C). Of these, Cyp4a10 and 14 are involved in microsomal oxidation of medium to long chain fatty acids specifically with a hydroxylated terminal ώ-carbon 32 . Upregulation of these Cyp proteins has not been previously reported in the liver though Cyp4a14 was elevated in the brains of PAHenu2 mice 33 . In the liver, Cyp4a10 and Cyp4a14 expression is known to be induced by a nuclear receptor peroxisome proliferator-activated receptor (PPARα), a ligand activated transcription factor that regulates lipid and lipoprotein metabolism. PPARα is mainly activated by elevated free fatty acids and upon activation it enhances hepatic lipid metabolism by upregulation of fatty acid translocase (FAT) /CD36 that functions to mediate the uptake of long chain fatty acids into the cell 32 . Fatty acids can subsequently be removed via increased peroxisomal and mitochondrial fatty acid beta-oxidation. Our data showed that the expression of both PPARα and FAT/CD36 were slightly increased in PAH enu2 mice suggestive of potential biological impact of Cyp4a10/Cyp4a14 upregulation (Fig. 5F). Substrates of Cyp4a10 (Cyp4a11 in humans), long chain poly unsaturated fatty acid (LC-PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to be low in PKU patients suggesting possible downstream consequences of increased Cyp4a10 expression 34 . In-fact lower levels of LC-PUFA are also observed in children on unrestricted diet suggesting that these changes are specific to the disease and not merely a consequence of PKU diet 35 . Besides potential microsomal omega oxidation via increased Cyp4a14, enzymes involved beta oxidation, Acox1 and Acot4, were also increased by proteomics. Elevation in Cyp4a14 has also been reported in models of nonalcoholic fatty liver disease and shown to contribute to hepatic steatosis and nonalcoholic steatohepatitis by increased oxidative stress due to lipid accumulation 32 . While we observed an increase in expression of various glutathione transferases (Fig. 6B, Supplementary Fig. S4C), enzymes that protect cell from reactive species, it should be noted that liver pathology indicative of massive oxidative stress or consistent elevation of liver enzymes was not observed in untreated PAH enu2 mice. Furthermore, neither liver pathology nor oxidative stress and elevated liver enzymes have been reported in PKU patients. However, PKU mice exhibit poor growth which can be corrected with Phe reduction suggesting Phe effect on energy metabolism and lipid dysfunction 36 . Taken together, our data highlights a novel observation of family of Cyp4a protein upregulation that may increase uptake of fatty acids and their oxidation. Whether this may contribute to lowered sera cholesterol, HDL and LDL observed in PKU patients is currently unclear.
It should be noted that the changes in cholesterol and lipid levels in our PAH enu2 mouse study were obtained with animals fed with normal rodent diet during their entire life span. However, understanding the causative nature of changes in lipid and cholesterol levels in PKU patients is clearly more complex as the disease pathology, compliance with Phe-restricted diet, fluctuations in Phe levels and other underlying genetic factors all likely impact lipid levels 30,[37][38][39] . It has been generally thought that the consumption of Phe-restricted diet consisting little animal derived products contributes to lower levels of lipids and cholesterol. However, even non-compliant PKU patients tend to have lower HDL levels 40 suggesting that lipid alterations in PKU patients are influenced by disease pathology in addition to diet. Interestingly, compliant PKU patients also tend to have high rate of being overweight thought to be at partially caused by the use of protein substitutes and commercial low-Phe products with high carbohydrate content 37,38 . However, the effect of Phe on lipid metabolism with increase fatty acid uptake and metabolism may also play a role. Lastly, the impact of lipid alterations and increased bodyweights in PKU patients on various co-morbidities such as cardiovascular disease and atherosclerosis are important topics of discussion for the care of PKU patients and should be aided with better understanding the underlying disease.
In summary, our data demonstrated that Phe reduction could effectively be obtained with PAH or PAL expression and resulted in improved brain health in mice. Obtaining normal brain Phe levels appeared to be especially critical for restoring neurotransmitter synthesis while amino acid transport was less critical. Our data also highlighted novel changes in lipid metabolism pathways in the PKU liver indicating elevated Phe has profound effects in other organs beyond the known neurotoxicity.

Materials and methods
Gene delivery vector generation. The expression cassette was based on liver-specific promoter mTTR482, hybrid intron, polyadenylation sites (bovine growth hormone [BGH]) and has been described before 41 . These expression elements were modified to contain A1MB2 enhancer (2 copies alpha1-microglobulin) upstream of mTTR482 and an intron consisting of CBA (chicken beta actin)/rabbit beta globin hybrid intron). The vectors encoded either human codon-optimized murine PAH or Anabaena variabilis PAL. Both proteins contained 3xFLAG fusion (DYKDDDDK) for detection purposes (PAH, N-terminal fusion and PAL, C-terminal fusion). Additionally, the PAL vector contained 0.9 kb A1AT filler sequence downstream of BGH pA. Plasmid vectors were confirmed for PAH and PAL production in vitro (data not shown). Single-stranded recombinant AAV vectors with AAV2 ITRs and a liver tropic AAV capsid were generated using triple transfection method followed by CsCl2 purification (Univ. Massachusetts Gene Therapy Core). Vector lots were quantitated by qPCR to BGHpA 41 .
Animal procedures. All animal procedures were approved by the Sanofi's Institutional Animal Care and Use Committee (IACUC) in an animal facility accredited by Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and in compliance with ARRIVE guidelines (http:// www. nc3rs. org. uk/ page. asp? id= 1357). A colony of PAH-deficient BTBR-PAH enu2 (PKU) mice was maintained at Taconic 42 . For both the studies (Fig. 1, Supplementary Fig. S1A), homozygous (n = 46-48) were divided into n = 8-10 per group and heterozygous male mice (n = 8-10) were obtained at 8-9 weeks of age and were housed in accordance with humane guidelines for animal care and use. Animals were individually caged and fed regular 16% protein diet. Vectors encoding PAH or PAL were administered by intravenous route via tail vein. For Phe measurement during the study, mice were anesthetized with isoflurane, blood was collected by retro-orbital sinus into EDTA collection tubes, spun and stored frozen until analysis. For termination, animals were euthanized humanely by CO 2 by asphyxiation as per Sanofi IACUC protocols. Some animals were intracardially perfused by PBS before tissue collection. Liver and brain samples were collected and frozen at -80 °C until or fixed in PFA for further analysis. Liver histopathology. Tissues were fixed in 10% neutral buffered formalin at the time of collection and processed as described in Singh et al. 29 The 5um paraffin embedded sections were stained with hematoxylin and eosin (H&E) were evaluated semi quantitatively for inflammatory cells (plasma cells, lymphocytes and rare macrophages) by a board-certified pathologist.

Serum chemistry analyses. Sera samples were analyzed for ALT and AST using the Randox Daytona
Clinical Chemistry analyzer. The results were obtained for ALT (kit catalog AL3875) and AST (kit catalog AS3876) by the Tris buffer without P5P 37 °C method for both analytes. This is a UV method that is used for the quantitative determination of these analytes. The principle of the AST reaction is α-oxoglutarate reacting with l-aspartate in the presence of AST that forms l-glutamate and oxaloacetate. The indicator reaction utilized the oxaloacetate for a kinetic determination of NADH consumption. The principle of the ALT reaction is α-oxoglutarate reacting with l-alanine in the presence of ALT that forms l-glutamate and pyruvate. The indicator reaction utilized the pyruvate for a kinetic determination of NADH consumption.
Nest building behavior assay. Nest building assay was adapted from Deacon et al. 44 with minor modifications described by Singh et al. 29 .
Gene expression of selected genes were validated by qPCR. The primer sequences used are listed in Supplemental Table S1.
Liver proteomics analysis. Liver samples were cut into pieces on dry ice and weighed. Then placed into pre-chilled Eppendorf tubes. Ten 2-mm zirconia beads were added to each frozen tissue on dry ice. Transfer tubes to wet ice and 10× w/v of water supplemented with Halt™ protease and phosphatase inhibitor cocktails (Cat. # 78440, Pierce Biotechnology, Rockford, IL) were added. The samples were homogenized in TissueLyser for 3 min at 30 Hz and at 4 °C. The homogenates were transferred to fresh, pre-chilled tube and the total protein contents were measured with BCA assay (BCA™ Protein Assay kit, Pierce Biotechnology, Rockford, IL). About 40 µg of each sample was transferred to protein lo-bind Eppendorf tubes, and then reduced, alkylated and digested with Lys-C (for overnight) followed by trypsin (for 2.5 h). RapiGest SF surfactant (Cat. # 186001861, Waters corporation, Milford, MA) was used to enhance protease digestion. The digestion was quenched with formic acid. The samples were incubated for 45 min at 37 °C and centrifuged at 22,000×g for 30 min at room temperature. The supernatants were transferred and were dried and re-suspended in 80 µl of 3% acetonitrile in 0.1% formic acid.
Mouse liver digests were analyzed by nanoACQUITY UPLC © (Waters Corporation, Milford, MA) coupled to Q Exactive™ High Field (HF)-X Hybrid Quadrupole-Orbitrap™ mass spectrometer (ThermoFisher Scientific). About 3 uL (~ 1.5 ug) digests from each sample were loaded onto a trap column (180 µm × 20 mm) from Waters Corporation with 5 µm 100 Å C18 medium and washed using a flow rate of 10 µl/min with 99% HPLC-grade water/1% acetonitrile (ACN)/0.01% formic acid (FA) for 3 min. Peptides were separated using a reversed phase nanoACQUITY UPLC 1.8 µm HSS T3 (100 µm × 100 mm) analytical column from Waters using a 65-min LC method at a flow rate of 500 nl/minute. Column temperature was maintained at 40 °C using column heater attached with UPLC © . MS data were acquired with data-dependent acquisition (DDA). using one full MS scan followed by twenty MS/MS scans. The full scan MS spectra were collected over 375-1600 m/z range with a maximum injection time of 30 ms, a resolution of 120,000 at 200 m/z and Automation Gain Control (AGC) target of 3e6. Fragmentation of precursor ions was performed by high-energy C-trap dissociation (HCD) with the normalized collision energy of 29 eV. MS/MS of the peptide ions were acquired at a resolution of 15,000 at 200 m/z and AGC target of 1e 5 where the spectra were collected at centroid mode.
Peptide/protein identification and quantification. Progenesis QI-P (Waters, Milford, MA) software and Scaffold (ver. 4, Proteome Software, Inc., Portland, OR) was used to analyze and align the DDA raw data files. The false discovery rate (FDR) was set to 1% at peptide precursor as well as protein level. Data generated from Progenesis QI-P in .csv format was imported into OmicSoft Studio transferred (QIAGEN) to process further for statistical analysis and visualization. Statistical analysis. Statistical analysis was performed using the GraphPad prism software version 8.02.
Blood Phe and Tyr (Fig. 1B,C, Supplementary Fig. S1A,B) were analyzed by Mixed-effects model followed by Tukey's multiple comparison. One-way ANOVA nonparametric Kruskal-Wallis test and Dunn's multiple comparison test was applied to the nesting assay (Fig. 4). The remaining data sets were analyzed using ordinary One-way ANOVA Tukey's multiple comparison.