An In-depth Comparison of the Pediatric and Adult Urinary N -Glycomes

We performed an in-depth characterization and comparison of the pediatric and adult urinary glycomes using a nanoLC-MS/MS based glycomics method, which included normal healthy pediatric (1-10 years, n=21) and adult (21-50 years, n=22) individuals. A total of 116 N-glycan compositions were identified, and 46 of them could be reproducibly quantified. We performed quantitative comparisons of the 46 glycan compositions between different age and sex groups. The results showed significant quantitative changes between the pediatric and adult cohorts. The pediatric urinary N-glycome was found to contain a higher level of high-mannose (HM), asialylated/afucosylated glycans (excluding HM), neutral fucosylated and agalactosylated glycans, and a lower level of trisialylated glycans compared to the adult. We further analyzed gender-associated glycan changes in the pediatric and adult group, respectively. In the pediatric group, there was almost no difference of glycan levels between males and females. In adult, the majority of glycans were more abundant in males than females, except the high-mannose, tetrasialylated and sulfated glycans. These findings highlight the importance to consider age-matching and adult sex-matching for urinary glycan studies. The identified normal pediatric and adult urinary glycomes can serve as a baseline reference for comparisons to other disease states affected by glycosylation.

The pooled and purified 2-AA labeled glycan samples were further methylamidated as previously described 26 with slight modifications. Briefly, the dried samples were dissolved in 50 μL of DMSO solution containing 2.5 M methylamine hydrochloride, followed by the addition of 25 μL of PyAOP (250 mM in 30% 4-methylmorpholine/DMSO). The reaction mixture was incubated at room temperature for 1 h with constant shaking, followed by purification. The purified glycans were dried in a vacuum centrifuge and stored at -20 °C prior to MS analysis.

Purification of Labeled N-glycans
The reaction mixture was dissolved in 1 mL of 80% ACN containing 50 mg of cellulose powder, followed by washing with 80% ACN. The labeled glycans were finally eluted after incubation with 50% ACN and dried prior to LC-MS analysis.

Nano-LC MS/MS Analysis of Labeled N-Glycans
The labeled N-glycans were resuspended in water and analyzed on a Q Exactive mass spectrometer (Thermo Scientific, Waltham, MA) equipped with a NanoLC 415 system (Eksigent, Dublin, CA). Glycans were separated by a ProteoPepII C18 column (New Objective) at 45°C. The mobile phases consisted of 0.1 % formic acid in water (Solvent A) and 0.1 % formic acid in 100 % ACN (Solvent B). The glycans were eluted using a gradient from 12 to 22% of mobile phase B over 33 min at 300 nL/min. Each sample was run in replicate. The mass spectrometer was operated in positive-ion mode with a spray voltage at 2.5 kV and capillary temperature at 300 °C. The full MS scans (m/z 400 to 2,000) were acquired at a resolution of 70,000 with automatic gain control (AGC) target of 3 × 10 6 ions and maximum ion transfer time (IT) of 20 ms. HCD MS/MS acquisitions were performed in a data-dependent mode, at a resolution of 35,000 at m/z 200. AGC target was 2 × 10 5 ions and maximum IT was 120 ms for MS/MS acquisitions and underfill ratio was 2.5%. The 6 most abundant precursor ions with charge state from 1 to 4 were selected for MS/MS. Precursor isolation window was 1.6 m/z. Monoisotopic precursor selection and dynamic exclusion (14s duration) were enabled. HCD fragmentation was with stepped normalized collision energy from 10-25%.

Data Analysis
by guest on November 5, 2020 Glycan identification was performed semi-manually assisted with our in-house software 27 . The compositions of N-glycans were assigned on the basis of accurate mass and MS/MS fragmentation. The theoretical mass for 2-AA labeled and methylamidated glycans were calculated as follows: the mass of modified glycans = the mass of unmodified glycans + the mass of methylamidated 2-AA (134.0844 for 12 C6-2-AA or 140.1045 for 13 C6-2-AA) + the number of sialic acids x 13.0316. Mass accuracy within 10 ppm was required for compositional assignment. The GlycoWorkbench 2.1 software 28 was additionally employed to assist in putative glycan structure annotation and in silico fragmentation analysis. Glycan quantification was performed semi-manually assisted with Xcalibur 3.0.63 (Thermo Fisher Scientific). The quantitative measurement was calculated based on the summed peak areas from extracted ion chromatographs (EIC) of the first three isotopic peaks of each glycan composition. If the glycan had multiple precursor ions, such as different charge states, the peak areas from all ions were added. For the relative quantification of each glycoform in the individual sample, the percentage of each glycoform towards the total glycome was calculated. Glycan compositions were abbreviated as follows: hexose (Hex), N-acetylhexosamine (HexNAc), fucose (Fuc), Nacetylneuraminic acid (NeuAc), and Sulfate (S).
The majority of urinary glycans were sialylated and fucosylated, and sulfated glycans were a minor species, accounting for approximately 1.0% of the total glycome. Since only the sialylated by guest on November 5, 2020 glycans were neutralized by methylamidation in this study, the percentages of negativelycharged sulfate glycans could be underestimated in this comparison.
Although we were not able to reveal complete structural information, partial structural information can be deduced based on a combination of accurate precursor masses, MS 2 fragment ions, known biosynthesis pathway and published structures. With the 2-AA label at the reducing end, it is easy to differentiate between fragment ions containing non-reducing and reducing end residues. A list of characteristic fragment ions resulting from glycosidic cleavages was generated, which allowed for rapid screening and identification of different glycan species (Table S2)

N-glycome from Healthy Human Urine is Age and Sex Dependent
In this study, we have included both pediatric (males: 1-10 years; female: 2-9 years) and adult groups (males: 21-50 years; female: 21-33 years). We identified a total of 116 glycan compositions. At the compositional level, there was no difference between the two age and sex groups. Among the total identified glycans, 46 of them were present across all the biological samples and technical replicates, and could be reproducibly quantified (coefficient of variation (CV) < 15% between technical replicates). These 46 glycans accounted for approximately 90% of the total glycome. We performed quantitative comparisons of the 46 glycan compositions between different age and sex groups, and 42 of them were up-or down-regulated (≥1.2 fold change, p<0.05) ( Table S3). These 42 glycans were divided into different groups based on their compositions and were further quantitatively compared based on age and sex, respectively.

Quantitative differences of N-glycans between Pediatric and Adult groups
We first compared the urinary glycan differences between the pediatric and adult groups. Table   2 shows glycan groups with significant changes (≥1.2 fold change, p<0.05). The results showed that total glycan level remain unchanged between the pediatric and adults. However, many groups of glycans sharing specific structural elements changed significantly. For example, highmannose, a-and mono-galactosylated, bisecting GlcNAc and neutral fucosylated glycans were by guest on November 5, 2020 all down-regulated, while trigalactosylated and trisialylated glycans were up-regulated in adults compared to the pediatric cohort.
Age-related changes were further analyzed in males and females separately, and the pattern of changes has similarities and showed the same change trend in both gender. It can be seen that the high-mannose, asialylated/afucosylated (excluding HM), neutral fucosylated and agalactosylated glycans were down-regulated while the trisialylated glycans were up-regulated in both adult males and adult females.

Sex Differences of N-glycans are Much Smaller in Pediatric than in Adult
We compared the glycan profiles between male and female groups, and found that males in general had higher abundance of glycans ( Table 3). We further analyzed gender-associated glycan changes in the pediatric and adult group respectively. In the pediatric group, there was almost no difference of glycan levels between males and females. In adult, the majority of glycans was more abundant in males than females, except high-mannose and tetrasialylated glycans.

DISCUSSION
This study reports 1.5 fold more glycan compositions than previously reported in human plasma using an LC-MS based method. 32 Among the 116 urinary glycan compositions identified in this study, 57 were shared with the published N-glycome of human serum/plasma 32,33 while half of them have not been reported in the serum/plasma. There were significant age-and genderassociated glycan differences in plasma, however such changes have not been studied in urine.
Most of the studies focused on the adult population 12,13,34,35 (age >20 years) while only one study focused on the younger population 14 (6-18 years). In this study, we are the first to have included both pediatric and adult urine, and found significant changes of the different glycan patterns between pediatric and adult cohorts and with similar change trend between genders.
We also found that sex differences of N-glycans were much smaller in pediatric than in adult cohort. Adult males had a higher abundance of glycans than adult females. The alteration of these glycan features could be caused by the changes in the expression level of their carrier by guest on November 5, 2020 glycoproteins and/or glycosyltransferases and glycosidases, and the sugar nucleotide donors involved in the glycosylation pathway. 36 The various urinary N-glycans found in this study are critical for many biological functions.
Those additional urinary N-glycans such as sulfated glycans not reported in serum may have been derived from urogenital system-originated glycoproteins. Sulfation is an N-glycan modification that is catalyzed by sulfotransferases. 37 Sulfate can be added to the core or the antennae of hybrid and complex N-glycan chains, and it has been found in a variety of urinary glycoproteins including uromodulin and podocalyxin. 38 It has been shown that the Sd a antigen can prevent the binding of NeuAc Similar to the plasma reports, we also found significant age-associated glycan changes in urine.
Our previous urinary proteome study 19 on healthy pediatric and adult males showed that the most abundant urinary protein, uromodulin, which carries around 30% high-mannose glycans, was down-regulated in the adult males. In addition, α-mannosidase II, which controls the conversion of high-mannose to complex N-glycans, was up-regulated in the adult male urinary proteome. Therefore, the decrease in high-mannose and increase in complex glycans (Trigalactosylated and Trisialylated glycans) we observed in this study could be partially due to the lower expression level of uromodulin and higher expression level of α-mannosidase II in the adult.
The increase of agalactosylated glycans with age has been frequently reported in the serum of adults older than 40 years 12,13,35 while the opposite change trend was reported in the age group of 6 to 18 years 14 . Our patient cohort was not designed to verify changes in middle age, but our by guest on November 5, 2020 results were consistent with the latter study in which we found less agalactosylated glycans in adults versus children. The decrease in a-and mono-galactosylated glycans and increase in trigalactosylated glycans could be a result of decreased activity of β-galactosidase or increased activity of β-1,4-galactosyltransferase (B4GALT) in the adult. It has been shown that plasmatic B4GALT activity exhibited a linear increase from infancy to centenarians. 46 The neutral fucosylated glycans were all found to be core-fucoyslated. Core-fucosylation, catalyzed by fucosyltransferase, FUT8, is vital for normal development and regulation of the immune system. About 70% of FUT8 knock-out mice died within three days after birth due to major defects in developmental growth and the respiratory system, while the survivors showed severe growth retardation and emphysema-like changes in the lungs. 47,48 Kreidberg et al.
reported that core fucosylation of α3β1 integrin plays a critical role in kidney and lung organogenesis. 49 Lack of core fucosylation can disrupt signaling mediated by epidermal growth factor receptor 50 and vascular endothelial growth factors. 51 The glycoprotein, epidermal growth factor (EGF), has the highest level in urine compared to other tissues and body fluids and is one of the more abundant urinary proteins. 52 EGF stimulates epithelial cell growth and metabolism 53 and is 9.8 times higher in the urine of pediatric males compared to adult males. 19 This suggests that increased urinary EGF could be one factor that contributes to the upregulation of the neutral fucosylated glycans and other glycans in the urine of children.
We found down-regulation of bisecting GlcNAc in female adults. Bisecting GlcNAc biosynthesis is catalyzed by the N-acetylglucosaminyltransferase, GnT-III, by introducing of GlcNAc in a β1-4 linkage to the mannose residue at the base of the trimannosyl core of the N-glycan. 54 Thus, the decreased bisecting GlcNAc in female adults could be due to the decreased activity of GnT-III.
Bisecting GlcNAc is expressed highly in the brain and kidney under normal condition 55,56 and it is involved in the maintenance of kidney homeostasis 57 and onset of Alzheimer's disease and/or progression in aging. 58 However, GnT-III-deficient mice were found to be viable and able to reproduce normally, suggesting that bisecting GlcNAc is dispensable for normal growth and development.

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Our results were consistent with the previous plasma N-glycome study in children and adolescents aged from 6 to 18 years which showed that sex differences are much smaller in children than in adults. 14 Shao et al 20

CONCLUSIONS
This is the first in-depth study of the normal pediatric and adult urinary glycomes. Our results demonstrated that urinary glycan composition is age independent while quantitatively is agedependent. In the meantime, urinary glycome is sex independent in the pediatric cohorts and sex-dependent in the adult cohorts. Age-and sex-specific quantitative differences between these glycomes further highlight the importance of understanding the variation among normal, i.e. non-diseased samples to better understand the urinary glycome. These findings also strongly emphasized the need to consider age matching and adult sex-matching for urinary by guest on November 5, 2020 https://www.mcponline.org Downloaded from glycan marker discovery. Based on previous reports, the reported differences between pediatric and adult samples could be due to complex underlying causes, including differential expression of a distinct class of glycoproteins, differential glycosidase and/or glycosyltransferase activity, and differences in growth and cell metabolism. The identified normal pediatric and adult urinary glycomes are helpful and highly impactful for future studies by serving as a baseline reference for comparisons to other disease states affected by glycosylation in both pediatric and adult populations.

Notes
The authors declare no competing financial interest.

DATA AVAILABILITY
All the data that support the findings of this study are available from the corresponding author on reasonable request. Glycomic LC-MS/MS raw data and all the Glycoworkbench annotated representative MS2 spectra (.