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Oligosaccharide analysis in urine by MALDI-TOF mass spectrometry for the diagnosis of lysosomal storage diseases.

The degradation of glycoproteins and glycolipids occurs predominantly in lysosomes, which contain a series of glycosidases and other hydrolases required for the complete degradation of the glycan components of glycoproteins or glycolipids. Deficiencies of lysosomal hydrolases, such as in lysosomal storage diseases (LSDs), (3) can lead to the accumulation of intermediate metabolites from glycoprotein or glycolipid degradation pathways, including free oligosaccharides (FOS) and glycoaminoacids, which may accumulate in urine. Increases in specific urinary FOS or glycoaminoacids are often indicative of specific lysosomal disorders.

A traditional method to analyze urinary oligosaccharide is 1-dimensional TLC, but this method has limited analytical specificity and sensitivity and provides no structural information for the oligosaccharides, which is often needed for diagnoses (1). Although TLC is the most widely used method for clinical testing of FOS in urine, other analytical techniques have been reported, and these techniques often require derivatization of reducing ends of FOS by a fluorophore or an ultraviolet-sensitive tag to achieve enough analytical sensitivity for analysis and detection by HPLC (2, 3). Analysis of FOS can also be achieved by using HPLC with mass spectrometry (MS) after derivatization by 3-methyl-1-phenyl-2-pyrazolin-5-1 or by 1-phenyl-3-methyl-5-pyrazolone (2, 4). One limitation of these methods is that they require a reducing end for derivatization and are not useful for the detection of glycoamino acids, which is necessary for the diagnosis of diseases such as aspartylglucosaminuria (AGU). A second limitation is that without permethylation to protect terminal sialic acid, the sialylated oligosaccharides are often not stable and are difficult to identify consistently. Finally, LC-MS has limited analytical sensitivity toward oligosaccharides with m/z >2000. Because oligosaccharides are difficult to ionize, a majority of the singly charged oligosaccharides with high molecular weight are missed by LC-MS. A MALDI-TOF method has also been described to analyze FOS after derivatization with 2-aminonaphthalene trisulfone (ANTS) (5). However, ANTS derivatization has limited analytical sensitivity because of the competition between the acidic character of the sulfone groups of ANTS and the basic properties of N-acetylglucosamine (GlcNAc) residues for ionization (6). In a recent multiinstitutional study comparing methods for profiling glycoprotein glycans, MALDI-time-of-flight/time-of-flight (MALDI-TOF/TOF) of permethylated oligosaccharides yielded adequate quantification and provided good correlation between different glycomics laboratories, which may be related to the fact that permethylation protects the weak terminal glycosidic linkage (e.g., sialic acid) (7). Here we describe a high-throughput screening method using MALDI-TOF/TOF analysis of permethylated urinary FOS and glycoaminoacids as a clinical tool for the diagnosis of oligosaccharidoses. A variety of urine samples from patients with different LSDs were analyzed and pathognomonic oligosaccharides or glycoaminoacids or their patterns were successfully identified for each condition.

Materials and Methods

MATERIALS

Iodomethane, anhydrous DMSO, 2,5-dihydroxybenzoic acid (DHB), sodium hydroxide, trifluoroacetic acid (TFA), and sodium acetate were purchased from Sigma-Aldrich. HPLC grade methanol, acetonitrile, and chloroform were purchased from Fisher Scientific. Sep-Pak C18 3cc Vac cartridges were purchased from Waters. Extra-Clean SPE Carbograph columns were purchased from Grace Davidson Discovery Science.

URINE SAMPLES

The Emory Genetics Laboratory is fully accredited by the College of American Pathologists and CLIA. Urine samples were from the collection of both Emory Genetics Laboratory and Greenwood Genetics Center as QC materials for routine clinical urinary FOS analysis by TLC. Control samples were categorized on the basis of age groups. This study was approved by the institutional review board and informed consent was obtained from all participants.

URINE SAMPLE PREPARATION

An appropriate volume of sample (to reflect a creatinine amount of 0.09 mg) was loaded to Sep-Pak C18 columns that were preactivated by methanol and then washed with 20 mL deionized water. The carbograph columns were activated by 3 mL 80:20 acetonitrile:water containing 0.1% TFA and then washed with 20 mL deionized water before loading. FOS bound on the carbograph columns were eluted off the columns with 4.5 mL from a mixture of 300 mL of acetonitrile and 700 mL deionized water containing 0.1% TFA. The eluents were lyophilized overnight at -20 [degrees]C to complete dryness.

PERMETHYLATION OF OLIGOSACCHARIDES

FOS were permethylated because the permethylation protects the terminal sialic acid residue and stabilizes the FOS during sample preparation and analysis (8, 9). Briefly, permethylation of oligosaccharides was performed as follows. Approximately 1 g of NaOH beads were grounded with 3 mL of anhydrous DMSO, then 0.4 mL of the DMSO/NaOH slurry and 0.1 mL of iodomethane were added to the dried FOS and the mixture was shaken vigorously for 1 h. The reaction was quenched by slow drop-wise addition of approximately 0.5 mL of deionized water with shaking between additions to lessen the effect of the highly exothermic reaction. Then 0.5 mL of chloroform was added and mixed thoroughly. The mixture was centrifuged at 1356g for 1 min to assist better separation of 2 layers. The upper aqueous layer was discarded and the lower chloroform layer was washed several times with dH2O until the water being removed was completely clear. The chloroform layer was dried under a gentle stream of nitrogen in a chemical hood.

The sample was redissolved in 500 xL of a mixture of 50 mL methanol and 50 mL deionized water and reloaded on a C18 column that was prewashed with 3 mL of methanol and 12-15 mL of dH2O. The FOS were eluted with a mixture of 50 mL methanol and 50 mL deionized water. The eluent was evaporated completely under speed-vac and resuspended in 50 xL of a mixture of 50 mL methanol and 50 mL deionized water.

INSTRUMENTATION AND ANALYSIS

The matrix solution was freshly prepared by dissolving 2.2 mg DHB in 0.2 mL from a mixture of 50 mL methanol and 50 mL deionized water and an 11-g/L sodium acetate solution was used to reach the final concentration of 1 mmol/L. A 0.5-xL sample of the permethylated urine oligosaccharide sample in a mixture of 50 mL methanol and 50 mL deionized water was spotted onto the MALDI plate and 0.5 xL of the matrix solution was loaded gently on the spotted sample. The sample-spotted plate was then left at room temperature for 10 min for drying. FOS analysis was carried out on a MALDI-TOF/TOF 4800 Analyzer (Applied Biosystems). The instrument was operated in positive reflector mode and laser power was set at 4880 (arbitrary units), digitizer 0.82 (arbitrary units). The processing method was the default positive reflector method. Each sample was run 3 times. We used the 4000 series explorer software (Applied Biosystems) to acquire the data. The data were analyzed by data explorer software (Applied Biosystems).

Results

The permethylated urinary FOS mass spectra from controls and from patients with different LSDs are described below. The permethylated FOS, such as those at m/z at 838.40,879.43,1240.61, and 1274.62, were present in almost every urine sample with a similar abundance pattern. Although we found a set of unique FOS present in the urine from patients with different lysosomal enzyme deficiencies, interestingly, among the patients we analyzed the relative abundance of these unique diagnostic FOS appeared different among patients with a different subtype of the disease, which are often defined by age of onset of symptoms.

CONTROLS

The permethylated FOS spectra of urine from control groups were similar between all age groups, with major ions at m/z 838.40, 879.43, 1240.61, and 1274.5, which correspond to various urinary FOS commonly found in healthy individuals (Table 1). We studied urine samples from 7 different age groups ranging from 0-1 months to [greater than or equal to] 17 years (0-1 months, 1-6 months, 6-24 months, 2-5 years, 5-9 years, 9-16 years, and [greater than or equal to] 17 years, n = 10 for each group). For the reference profiles from children [less than or equal to] 1 month old, there were increased fucosylated FOS at m/z 651.32, 825.86, 1070.37, 1100.4, 1550.58, 1723.0, and 1897.0. In addition, Hexose3 at m/z 681.71 and Hexose3HexNAc1 at m/z 926.4 were also increased in very young infants (Fig. 1A). The relative intensities of these major peaks were very similar among all the reference groups with age >1 month (Fig. 1B). We suspect that the presence of fucosylated and small polyhexose oligosaccharides could be related to immature intestinal function in very young infants.

GM1 GANGLIOSIDOSIS

The representative profile of permethylated FOS from patients with GM1 gangliosidosis (n = 10) had a mass spectrum with major ions at m/z, 1171.58, 1375.68, 1620.81,1824.91, and 2274.14. The galactosylated FOS corresponding to these ions are listed in Table 1, and the spectrum shown in Fig. 2, A and B. The increases in galactosylated FOS are characteristic of GM1 gangliosidosis due to the deficiency of [beta]-galactosidase, as described previously (10). Interestingly, we found that the increases in these galactosylated urinary FOS were higher in the patients (n = 2) with early onset of GM1 gangliosidosis (disease onset age <1 year) (Fig. 2A) compared to the patients (n = 8) with mild juvenile onset (disease onset age >2 years) (Fig. 2B). For example, the ratio of a galactosylated oligosaccharide at m/z 1171 with a normal oligosaccharide at m/z 879 was at 1.4 in early-onset GM1 gangliosidosis, whereas it was 1.1 in a patient with later-onset GM1 gangliosidosis. The triantennary galactosylated FOS at m/z 2274 presented only in the urine from patients with early onset of the disease, but not in the urine from any patient with later onset. Interestingly, there were no detectable differences in these patients with regard to the [beta]-galactosidase activities in leukocytes (data not shown).

ASPARTYLGLUCOSAMINURIA

Aspartylglucosaminidase is a lysosomal hydrolase that cleaves the bond between asparagine (Asn) and GlcNAc and is involved in the degradation of glycoproteins within lysosomes. A genetic deficiency of aspartylglucosaminidase causes the accumulation of glycoaminoacids, e.g., GlcNAc-Asn and other glycoproteins in lysosomes, leading to AGU (11, 12). The representative urinary FOS spectra from the patients (n = 4) with AGU revealed major diagnostic permethylated glycoamino acids at m/z 629.1, 990.28, 1078.01, and 1439.43, with the predicted composition and structure shown in Table 1. All peaks corresponded to oligosaccharides with Asn attached to sugars. The concentrations of these 4 glycoaminoacids were higher in patients with early-onset AGU (n = 1) (Fig. 2C) compared to those with late-onset AGU (n = 3) (Fig. 2D). However, we must note that there we analyzed the urine from only 1 patient with early-onset disease. For example, the glycoamino acid at m/z 629 and its ratio with normal FOS at m/z 879 was 1.3 in the patient with early-onset disease and 0.2 in the patients with later-onset disease. Although 2 of these glycoamino acids, at m/z 990.28 and 1078.01, can be present in very small amounts in some controls, the observed increase of all 4 glycoaminoacids was found to be diagnostic for AGU. The detailed molecular structures of the glycoamino acids representing these ions are shown in Fig. 2, C and D, and in Table 1.

MUCOLIPIDOSIS II AND III

Mucolipidosis (ML) types II and III are caused by a deficiency of GlcNAc phosphotransferase (GNPT), which creates the mannose-6-phosphate lysosomal targeting signal, and is associated with multiple lysosomal hydrolase deficiencies. MLII and MLIII are classified according to the clinical severity of the disease. The early-onset and severe form of GNPT deficiency is called MLII, and the late-onset and milder form MLIII. The representative mass spectra of urinary FOS from patients with ML (n = 10) was characterized by the increases in FOS with a terminal mannose, sialic acid, or galactose at m/z 722.76, 1532.76, 1736.86, 1824.88, 2186.08, and 2547.26 (Table 1), consistent with defects in multiple lysosomal hydrolases, because sialidase, [beta]-galactosidase, and a-mannosidase are hydrolases that use phosphorylated mannose generated by GNPT as their lysosomal targeting signal (Fig. 2, E and F, and Table 1). The relative intensity of these different FOS, particularly the sialylated glycans at m/z 1532.76 and 2547.26, were higher in MLII, with their 879 ratio at 0.8 and 0.3 compared to the MLIII profile, with their 879 ratio at 0.15 and 0.04, respectively (Fig. 2, E and F). Urine samples from 5 patients with MLII and 5 patients with MLIII, known to have mutations in GNPTAB4 (N-acetylglucosamine-1 -phosphate transferase, alpha and beta subunits), were classified by their age of onset of symptoms. Clinical symptoms of MLII patients are apparent from early infancy or prenatally, whereas in MLIII patients symptoms present in childhood with slow progression. In patients with the clinical diagnosis of MLII, the increases of multiantennary sialylated glycans, such as the oligosaccharide at m/z 3357.66, were present, whereas these triantennary oligosaccharides were absent in urinary FOS from patients with MLIII.

GALACTOSIALIDOSIS

Galactosialidosis is an autosomal recessive genetic disorder caused by a primary defect of the CTSA gene (cathepsin A) (chromosomal locus, 20ql3.1) (13). Galactosialidosis is due to combined cellular deficiency of [beta]-galactosidase and neuraminidase (14). The representative mass spectrum of urinary oligosaccharides from patients (n = 3) with galactosialidosis was characterized by FOS at m/z 1532.76, 1824.91, 2186.08, 2547.26, 2996.48, and 3357.66. All these FOS correspond to both sialylated and galactosylated FOS, and the biantennary complex-type disialo oligosaccharide at m/z 2547 appeared to be the most dominant oligosaccharide in a severe form of galactosialidosis (n = 1) (Fig. 2G), whereas the milder form (n = 2) (Fig. 2H) appeared to have a similar profile to MLII except for concentrations of high-mannose oligosaccharides that were within reference intervals. In particular, the oligosaccharide at m/z 722.36, predicted to be Man2GlcNAc1, was not increased in galactosialidosis urine but was increased in MLII or MLIII. Note that we analyzed urine from only 1 patient with the severe form of galactosialidosis. Although sialylated FOS are prominent in urine from patients with galactosialidosis, there was no presence of polysialylated FOS, which differed from urinary FOS profiles of patients with sialidosis.

FUCOSIDOSIS

Fucosidosis is a very rare autosomal-recessive LSD due to the deficiency of [alpha]-L-fucosidase. Type I fucosidosis is a severe form with neonatal onset, and type II is a milder subtype (15). The spectra of permethylated FOS of 2 patients (n = 2) with fucosidosis disorder (1 with type I and 1 with type II) were characterized by monoor difucosylated oligosaccharides at m/z 1053.62, 1346.38,1550.58,1875.90, and 2173.21, with predicted compositions shown in Table 1. The small fucosylated glycoaminoacid at m/z 599.0 is also specific for fucosidosis and has been reported previously in patients with type I fucosidosis; however, this peak was not present in our patient with type II fucosidosis (Fig. 2, I and J, and Table 1). Based on our observation for only 2 known patients, 1 with a severe (type I) and 1 with a mild late-onset (type II) profile, the relative abundance of fucosylated oligosaccharides did not appear to correlate with clinical severity in fucosidosis. However, as previously reported, the presence of fucosylated glycoaminoacids appears to differentiate the type I from the type II [alpha]-fucosidosis.

POMPE DISEASE

Pompe disease, also known as GSDII (glycogen storage disease type II) or acid maltase deficiency (MIM 232300), is an autosomal recessive neuromuscular disorder caused by mutations in the gene that encodes the lysosomal hydrolase acid a-glucosidase (GAA; EC 3.2.1.20) (16). In Pompe disease, the severity of the disease is primarily related to the extent of glycogen deposition and the subsequent damage to affected tissues. The glucose tetrasaccharide Glc4 (Glc[alpha]1-6Glc[alpha]]1-4Glc[alpha] 1-4Glc) was reported to be present in the urine of a patient with Pompe disease. In addition, it is often used as a diagnostic marker and a noninvasive means of assessing glycogen storage and disease progression as well as for monitoring of enzyme replacement therapy (17).

The oligosaccharide profile of urine from patients with Pompe disease (n = 11) by the MALDITOF method was characterized by increases in multiple ions such as m/z 885.43, 1089.53, 1293.63, 1497.73, and 1701.83, representing polyhexose (assumed to be polyglucose) from Hexose4 to Hexose8 (Fig. 3A and Table 1).

GAUCHER DISEASE

Gaucher disease, the most common LSD, arises from the inherited deficiency of the lysosomal enzyme glucocerebrosidase/[beta]-glucocerebrosidase, which results in the accumulation of glycolipid compound in cells. There are 3 types. Type 1 is nonneuronopathic with primarily visceral signs and symptoms that range widely in severity. Infantile-onset type 2 and later-onset type 3 involve the central nervous system. In Gaucher disease, there is an imbalance in the monocyte-macrophage system between the rates of formation and degradation of glucosylceramide due to low activity of the lysosomal enzyme [beta]-glucocerebrosidase, resulting in hepatomegaly, splenomegaly, and bone and lung pathology (18,19). The representative urinary oligosaccharide profile of patients with Gaucher disease (n = 11) was characterized by the increase in multiple high-mannose-type oligosaccharides (from Man2GlcNAc1 to Man6GlcNAc1) as previously described (2, 20), including ions at m/z 722.36, 926.46, 1130.56, 1334.66, and 1538.76 and subtle increases of sialylated FOS with m/z 1532.76,1736.86,2186.3, and 2547.6 (Fig. 3B). The underlying mechanism of accumulation of high-mannose FOS species in Gaucher diseases has not been fully studied and we have not observed any correlations be tween these FOS biomarkers and the clinical phenotypes of the patients.

[alpha]-mannosidosis

[alpha]-mannosidosis is caused by mutations in the mannosidase, alpha, class 2B, member 1 (MAN2B1) gene (MIM 609458) located on chromosome 19 (19p13.2q12), coding for the intracellular enzyme amannosidase. Lysosomal accumulation of mannoserich oligosaccharides results from the deficiency of a-mannosidase, which is also excreted in the urine (21-25). The mass spectrum of the permethylated oligosaccharides of patients (n = 4) with [alpha]-mannosidosis was distinguished by major ions at m/z 722.36, 926.46, 1130.56, 1334.66, 1538.76, 1742.86, 1946.96, and 2151.06. These ions primarily correspond to different numbers of mannose and 1 GlcNAc at the reducing end (Fig. 3C and Table 1). The major difference in FOS profiles between [alpha]-mannosidosis and Gaucher disease was the presence of Man7-Man9GlcNAc1 species at m/z 1742.86,1946.96, and 2151.06, respectively, in the [alpha]-mannosidosis profile but absent in the Gaucher disease profile (Fig. 3C). Overall, patients (n = 2) with early onset of [alpha]-mannosidosis accumulated more mannose FOS in urine than patients (n = 2) with very mild clinical presentation. However, the pattern was not as distinct as other oligosaccharidosis profiles shown in Fig. 2.

GM2 GANGLIOSIDOSIS

GM2 gangliosidosis (OMIM No. 230700) is a rare and heterogeneous autosomal recessive LSD caused by the deficiency of [beta]-hexosaminidase activities due to mutations in either HEXA [hexosaminidase A (alpha polypeptide)] (15q23-q24) or HEXB [hexosaminidase B (beta polypeptide)] (5q13) or deficiency of the noncatalytic GM2 activator. Mutations in HEXB cause a combined deficiency of [beta]-hexosaminidases A and B, which is categorized as Sandhoff disease (OMIM no. 268800). [beta]-Hexosaminidases A and B are involved not only in the removal of the [beta]-N-acetylgalactosamine residues from the nonreducing terminal of the oligosaccharide of GM2 ganglioside, but also in the removal of GlcNAc in glycoprotein degradation (26-28). A representative oligosaccharide profile of a urine sample from an infant with Sandhoff disease is shown as Fig. 3E. A characteristic ion pattern for this condition (n = 2) included ions at m/z 967.43, 1212.58, 1416.65, 1661.76, and 1906.87. All of these ions are from oligosaccharides with terminal GlcNAc with the predicted structures shown in Fig. 3E and Table 1.

SIALIDOSIS

Sialidosis is an LSD caused by the deficiency of NEU1 (a-N-acetyl neuraminidase-1) (lysosomal sialidase, EC 3.2.1.18) (29), encoded by the gene NEU1 [sialidase 1 (lysosomal sialidase)]. Sialidosis is characterized by the progressive lysosomal accumulation of sialylated glycopeptides and oligosaccharides. It is classified into 2 main clinical variants, termed type I and II. Type I is the milder form of the disease and occurs as a late-onset, normosomatic form of the disorder. Type II, which can in turn be subdivided into 3 forms (congenital, infantile, and juvenile) (29, 30), is a severe early-onset form. Sialidosis (n = 1) can be easily recognized using the MS analysis of permethylated FOS with characteristic peaks at m/z 1532.76, 1578.66, 1614.72, 2547.26, 2629.16, and 2711.02, all of which are FOS with a terminal sialic acid (Fig. 3D). The presence of the polysialylated FOS at m/z 1578.66 is diagnostic for sialidosis and differentiates it from galactosialidosis, In addition, FOS at m/z 1614.72 (Sial1Hexose1HexNAc4) and m/z 2629.16 (Sial2Hexose3HexNAc1) appeared more prominent even in later-onset sialidosis (n = 2) compared to their concentrations in a patient with neonatal onset of galactosialidosis.

Discussion

In this study, we have shown that different lysosomal disorders can be identified by a unique pattern of various FOS that arise from incomplete degradation related to enzyme deficiencies or possibly because of incomplete biosynthesis. The oligosaccharide analysis by MALDI-TOF covers a wide molecular range from m/z 300 to 4000, including most of the known urinary FOS. With this mass information we could predict the compositions of the oligosaccharides and the specific patterns due to the deficiency of particular enzymes. This unique feature enabled us to diagnose different lysosomal diseases with this method. The advantage of the method described here over previously reported electrospray ionization-MS methods is that it provides more stable FOS profiles and more analytical sensitivity at the higher m/z range and can also detect glycoaminoacids, which are important for the diagnosis of AGU (2, 4). The assay identified 43 characteristic FOS combinations, which provide diagnostic patterns for each of the 10 lysosomal storage conditions described here. In addition, our assay was able to characterize specific patterns of subtypes of some oligosaccharidosis conditions and differentiate between the subtypes with severe early onset and mild clinical presentation. This technique can also be used to screen numerous other disorders characterized by urinary excretion of oligosaccharides, such as Pompe disease and CDG-IIb (congenital disorder of glycosylation IIb) (31). Compared to previous reports of FOS analysis in oligosaccharidosis, this study provides the most comprehensive profiling for each condition.

In summary, urinary high-resolution oligosaccharide analysis by MALDI-TOF/TOF MS after permethylation enabled us to screen for most known oligosaccharidosis associated with a wide variety of clinical severities and is potentially a reliable clinical tool for the first-tier screening of different LSDs.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, oranalysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: M. He, Clinical Education and Test Translation (CETT) program, a component of the NIH Office of Rare Diseases Research (ORDR).

Expert Testimony: None declared.

Patents: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We are grateful to Dr. Tim Wood from Greenwood Genetic Center for providing us samples from known patients with oligosaccharidoses.

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Baoyun Xia, [1], ([dagger]) Ghazia Asif, [1], ([dagger]) Leonard Arthur, [1] Muhammad A. Pervaiz, [1] Xueli Li, [1] Renpeng Liu, [2] Richard D. Cummings, [2] and Miao He [1] *

[1] Department of Human Genetics, 2 Department of Biochemistry and the Glycomics Center, Emory University, Atlanta, GA.

([dagger]) Baoyun Xia and Ghazia Asif contributed equally to the work, and both should be considered as first authors.

* Address correspondence to this author at: Department of Human Genetics, Emory University, 2165 N. Decatur Rd., Decatur, GA, 30033. Fax 404-778-8559;

e-mail miao.he@emory.edu.

Received January 4, 2013; accepted April 22, 2013.

Previously published online at DOI: 10.1373/clinchem.2012.201053

[3] Nonstandard abbreviations: LSD, lysosomal storage disease; FOS, free oligosaccharides; MS, mass spectrometry; AGU, aspartylglucosaminuria; ANTS, 2-aminonaphthalene trisulfone; GlcNAc, N-acetylglucosamine; MALDI-TOF/TOF, MALDI-time-of-flight/time-of-flight; DHB, 2,5-dihydroxybenzoic acid; TFA, trifluoroacetic acid; Asn, asparagine; ML, mucolipidosis; GNPT, GlcNAc phosphotransferase.

Table 1. Urinary oligosaccharides identified in controls and in the
patients with different genetic diseases.

m/z       Predicted     Predicted components      Control      GM1
          structure                               (n = 30)   (n = 10)
             (a)

599.0                 Fucosel HexNAc1-Asn
629.1                 Hexosel HexNAcI-Asn
651.32                Hexose2Fuc1                 +
681.33                Flexose3                    +
692.35                Flexosel FlexNAcI Fud       +/-
722.36                Flexose2FlexNAc1            +/-
825.86                Flexose2Fuc2                +/-
838.42                Neu5Ac1 Flexose2            +          +
879.43                Neu5Ac1 Flexosel FlexNAcI   +          +
885.43                Flexose4                    +/-
926.46                Flexose3 FlexNAcI           +/-
990.28                Neu5Ac1 Flexosel
                        FlexNAcI-Asn
967.43                Flexose2FlexNAc2            +/-
1053.62               Neu5Ac1 Flexosel
                        FlexNAcI Fud
1078.01               Flexose2 FlexNAc2-Asn       +/-
1089.53               Flexose5                    +/-
1130.56               Flexose4 FlexNAcI           +/-
1171.58               Flexose3FlexNAc2            +/-        + + +
1212.58               Flexose2FlexNAc3
1240.61               Neu5Ac2 Flexosel FlexNAcI   +          +
1274.62               Flexose3FlexNAc1 Fuc3       +          +
1293.63               Flexose6                    +/-
1334.66               Flexose5FlexNAc1
1346.38               Fucosel Flexose3FlexNAc2
1375.68               Flexose4FlexNAc2                       + + +
1416.65               Flexose3FlexNAc3
1439.43               Neu5Ac1 Flexose2Flex-
                        NAc2-Asn
1497.73               Flexose7
1532.76               Neu5Ac1 Hexose3HexNAc2
1538.76               Hexose6HexNAc1
1550.58               Fucosel Hexose4HexNAc2
1578.66               Sial3HexoseHexNAc1
1614.72               Siall Hexosel HexNAc4
1620.81               Hexose 4HexNAc3                        + +
1661.76               Hexose3HexNAc4
1701.83               Hexose8
1736.86               Neu5Ac1 Hexose4HexNAc2
1742.86               Hexose7HexNAc1
1824.91               Hexose5HexNAc3                         + +
1875.90               Hexose8Fuc1
1881.71               Neu5Ac1 Flexose2FlexNAc2
1906.85               Flexose3FlexNAc5
1946.96               Flexose8FlexNAc1
1981.98               Neu5Ac1 Flexose4FlexNAc3
2151.06               Flexose9FlexNAc1
2173.21               Flexose5 FlexN Ac3 Fuc2
2186.08               Neu5Ac1 Flexose5FlexNAc3               +
2274.14               Flexose6FlexNAc4                       + +
2343.16               Neu5Ac2 Flexose4FlexNAc3
2547.26               Neu5Ac2 Hexose5 HexN Ac3
2629.16               Neu5Ac2 Hexose3 HexNAc5
2996.48               Neu5Ac2 Hexose6HexNAc4                 +
3357.66               Neu5Ac3Hexose6HexNAc4

m/z       Predicted     Predicted components        AGU      ML II or
          structure                               (n = 4)     ML III
             (a)                                             (n = 10)

599.0                 Fucosel HexNAc1-Asn
629.1                 Hexosel HexNAcI-Asn         + +
651.32                Hexose2Fuc1
681.33                Flexose3
692.35                Flexosel FlexNAcI Fud
722.36                Flexose2FlexNAc1                       + + +
825.86                Flexose2Fuc2
838.42                Neu5Ac1 Flexose2            +          +
879.43                Neu5Ac1 Flexosel FlexNAcI   +          +
885.43                Flexose4
926.46                Flexose3 FlexNAcI
990.28                Neu5Ac1 Flexosel            + + +
                        FlexNAcI-Asn
967.43                Flexose2FlexNAc2
1053.62               Neu5Ac1 Flexosel
                        FlexNAcI Fud
1078.01               Flexose2 FlexNAc2-Asn       + + +
1089.53               Flexose5
1130.56               Flexose4 FlexNAcI
1171.58               Flexose3FlexNAc2                       + +
1212.58               Flexose2FlexNAc3
1240.61               Neu5Ac2 Flexosel FlexNAcI   +          +
1274.62               Flexose3FlexNAc1 Fuc3       +          +
1293.63               Flexose6
1334.66               Flexose5FlexNAc1
1346.38               Fucosel Flexose3FlexNAc2
1375.68               Flexose4FlexNAc2
1416.65               Flexose3FlexNAc3
1439.43               Neu5Ac1 Flexose2Flex-       + +
                        NAc2-Asn
1497.73               Flexose7
1532.76               Neu5Ac1 Hexose3HexNAc2                 + + +
1538.76               Hexose6HexNAc1
1550.58               Fucosel Hexose4HexNAc2
1578.66               Sial3HexoseHexNAc1
1614.72               Siall Hexosel HexNAc4       +/-        +/-
1620.81               Hexose 4HexNAc3
1661.76               Hexose3HexNAc4
1701.83               Hexose8
1736.86               Neu5Ac1 Hexose4HexNAc2                 +
1742.86               Hexose7HexNAc1
1824.91               Hexose5HexNAc3              +          + +
1875.90               Hexose8Fuc1
1881.71               Neu5Ac1 Flexose2FlexNAc2
1906.85               Flexose3FlexNAc5
1946.96               Flexose8FlexNAc1
1981.98               Neu5Ac1 Flexose4FlexNAc3    +          + +
2151.06               Flexose9FlexNAc1
2173.21               Flexose5 FlexN Ac3 Fuc2
2186.08               Neu5Ac1 Flexose5FlexNAc3    + +        + +
2274.14               Flexose6FlexNAc4
2343.16               Neu5Ac2 Flexose4FlexNAc3               +/-
2547.26               Neu5Ac2 Hexose5 HexN Ac3    + +        + + +/+ +
2629.16               Neu5Ac2 Hexose3 HexNAc5                +/-
2996.48               Neu5Ac2 Hexose6HexNAc4      +/-        + +
3357.66               Neu5Ac3Hexose6HexNAc4       +/-        + +

m/z       Predicted     Predicted components      Galactosialidosis
          structure                               (n = 3)
             (a)

599.0                 Fucosel HexNAc1-Asn
629.1                 Hexosel HexNAcI-Asn
651.32                Hexose2Fuc1
681.33                Flexose3
692.35                Flexosel FlexNAcI Fud
722.36                Flexose2FlexNAc1
825.86                Flexose2Fuc2
838.42                Neu5Ac1 Flexose2            +
879.43                Neu5Ac1 Flexosel FlexNAcI   +
885.43                Flexose4
926.46                Flexose3 FlexNAcI
990.28                Neu5Ac1 Flexosel
                        FlexNAcI-Asn
967.43                Flexose2FlexNAc2
1053.62               Neu5Ac1 Flexosel
                        FlexNAcI Fud
1078.01               Flexose2 FlexNAc2-Asn
1089.53               Flexose5
1130.56               Flexose4 FlexNAcI
1171.58               Flexose3FlexNAc2
1212.58               Flexose2FlexNAc3
1240.61               Neu5Ac2 Flexosel FlexNAcI   +
1274.62               Flexose3FlexNAc1 Fuc3       +
1293.63               Flexose6
1334.66               Flexose5FlexNAc1
1346.38               Fucosel Flexose3FlexNAc2
1375.68               Flexose4FlexNAc2
1416.65               Flexose3FlexNAc3
1439.43               Neu5Ac1 Flexose2Flex-
                        NAc2-Asn
1497.73               Flexose7
1532.76               Neu5Ac1 Hexose3HexNAc2      + + + +
1538.76               Hexose6HexNAc1
1550.58               Fucosel Hexose4HexNAc2
1578.66               Sial3HexoseHexNAc1
1614.72               Siall Hexosel HexNAc4
1620.81               Hexose 4HexNAc3
1661.76               Hexose3HexNAc4
1701.83               Hexose8
1736.86               Neu5Ac1 Hexose4HexNAc2
1742.86               Hexose7HexNAc1
1824.91               Hexose5HexNAc3              + +
1875.90               Hexose8Fuc1
1881.71               Neu5Ac1 Flexose2FlexNAc2
1906.85               Flexose3FlexNAc5
1946.96               Flexose8FlexNAc1
1981.98               Neu5Ac1 Flexose4FlexNAc3    + +
2151.06               Flexose9FlexNAc1
2173.21               Flexose5 FlexN Ac3 Fuc2
2186.08               Neu5Ac1 Flexose5FlexNAc3    + +
2274.14               Flexose6FlexNAc4
2343.16               Neu5Ac2 Flexose4FlexNAc3    +/-
2547.26               Neu5Ac2 Hexose5 HexN Ac3    ++++/+++
2629.16               Neu5Ac2 Hexose3 HexNAc5     +/-
2996.48               Neu5Ac2 Hexose6HexNAc4      + +
3357.66               Neu5Ac3Hexose6HexNAc4       + +

m/z       Predicted     Predicted components      Fucosidosis
          structure                               (n = 2)
             (a)

599.0                 Fucosel HexNAc1-Asn         + +/- (b)
629.1                 Hexosel HexNAcI-Asn
651.32                Hexose2Fuc1
681.33                Flexose3
692.35                Flexosel FlexNAcI Fud       + +
722.36                Flexose2FlexNAc1
825.86                Flexose2Fuc2                + +
838.42                Neu5Ac1 Flexose2            +
879.43                Neu5Ac1 Flexosel FlexNAcI   +
885.43                Flexose4
926.46                Flexose3 FlexNAcI
990.28                Neu5Ac1 Flexosel
                        FlexNAcI-Asn
967.43                Flexose2FlexNAc2
1053.62               Neu5Ac1 Flexosel            + + +
                        FlexNAcI Fud
1078.01               Flexose2 FlexNAc2-Asn
1089.53               Flexose5
1130.56               Flexose4 FlexNAcI
1171.58               Flexose3FlexNAc2
1212.58               Flexose2FlexNAc3
1240.61               Neu5Ac2 Flexosel FlexNAcI   +
1274.62               Flexose3FlexNAc1 Fuc3       +
1293.63               Flexose6
1334.66               Flexose5FlexNAc1
1346.38               Fucosel Flexose3FlexNAc2    + + + +
1375.68               Flexose4FlexNAc2
1416.65               Flexose3FlexNAc3
1439.43               Neu5Ac1 Flexose2Flex-
                        NAc2-Asn
1497.73               Flexose7
1532.76               Neu5Ac1 Hexose3HexNAc2
1538.76               Hexose6HexNAc1
1550.58               Fucosel Hexose4HexNAc2      + + +
1578.66               Sial3HexoseHexNAc1
1614.72               Siall Hexosel HexNAc4
1620.81               Hexose 4HexNAc3
1661.76               Hexose3HexNAc4
1701.83               Hexose8
1736.86               Neu5Ac1 Hexose4HexNAc2
1742.86               Hexose7HexNAc1
1824.91               Hexose5HexNAc3
1875.90               Hexose8Fuc1                 + +
1881.71               Neu5Ac1 Flexose2FlexNAc2
1906.85               Flexose3FlexNAc5
1946.96               Flexose8FlexNAc1
1981.98               Neu5Ac1 Flexose4FlexNAc3
2151.06               Flexose9FlexNAc1
2173.21               Flexose5 FlexN Ac3 Fuc2     + +
2186.08               Neu5Ac1 Flexose5FlexNAc3
2274.14               Flexose6FlexNAc4
2343.16               Neu5Ac2 Flexose4FlexNAc3
2547.26               Neu5Ac2 Hexose5 HexN Ac3
2629.16               Neu5Ac2 Hexose3 HexNAc5
2996.48               Neu5Ac2 Hexose6HexNAc4
3357.66               Neu5Ac3Hexose6HexNAc4

m/z       Predicted     Predicted components      Sialidosis
          structure                               (n = 3)
             (a)

599.0                 Fucosel HexNAc1-Asn
629.1                 Hexosel HexNAcI-Asn
651.32                Hexose2Fuc1
681.33                Flexose3
692.35                Flexosel FlexNAcI Fud
722.36                Flexose2FlexNAc1
825.86                Flexose2Fuc2
838.42                Neu5Ac1 Flexose2            +
879.43                Neu5Ac1 Flexosel FlexNAcI   +
885.43                Flexose4
926.46                Flexose3 FlexNAcI
990.28                Neu5Ac1 Flexosel
                        FlexNAcI-Asn
967.43                Flexose2FlexNAc2
1053.62               Neu5Ac1 Flexosel
                        FlexNAcI Fud
1078.01               Flexose2 FlexNAc2-Asn
1089.53               Flexose5
1130.56               Flexose4 FlexNAcI
1171.58               Flexose3FlexNAc2
1212.58               Flexose2FlexNAc3
1240.61               Neu5Ac2 Flexosel FlexNAcI   +
1274.62               Flexose3FlexNAc1 Fuc3       +
1293.63               Flexose6
1334.66               Flexose5FlexNAc1
1346.38               Fucosel Flexose3FlexNAc2
1375.68               Flexose4FlexNAc2
1416.65               Flexose3FlexNAc3
1439.43               Neu5Ac1 Flexose2Flex-
                        NAc2-Asn
1497.73               Flexose7
1532.76               Neu5Ac1 Hexose3HexNAc2      + + + +
1538.76               Hexose6HexNAc1
1550.58               Fucosel Hexose4HexNAc2
1578.66               Sial3HexoseHexNAc1          +/-
1614.72               Siall Hexosel HexNAc4       + + +
1620.81               Hexose 4HexNAc3
1661.76               Hexose3HexNAc4
1701.83               Hexose8
1736.86               Neu5Ac1 Hexose4HexNAc2
1742.86               Hexose7HexNAc1
1824.91               Hexose5HexNAc3
1875.90               Hexose8Fuc1
1881.71               Neu5Ac1 Flexose2FlexNAc2    +
1906.85               Flexose3FlexNAc5
1946.96               Flexose8FlexNAc1
1981.98               Neu5Ac1 Flexose4FlexNAc3    +
2151.06               Flexose9FlexNAc1
2173.21               Flexose5 FlexN Ac3 Fuc2
2186.08               Neu5Ac1 Flexose5FlexNAc3    +
2274.14               Flexose6FlexNAc4
2343.16               Neu5Ac2 Flexose4FlexNAc3    + +

2547.26               Neu5Ac2 Hexose5 HexN Ac3    + + +
2629.16               Neu5Ac2 Hexose3 HexNAc5     + + +
2996.48               Neu5Ac2 Hexose6HexNAc4      +
3357.66               Neu5Ac3Hexose6HexNAc4       +

m/z       Predicted     Predicted components      Gaucher
          structure                               disease
             (a)                                  (n = 11)

599.0                 Fucosel HexNAc1-Asn
629.1                 Hexosel HexNAcI-Asn
651.32                Hexose2Fuc1
681.33                Flexose3
692.35                Flexosel FlexNAcI Fud
722.36                Flexose2FlexNAc1            + + +
825.86                Flexose2Fuc2
838.42                Neu5Ac1 Flexose2            +
879.43                Neu5Ac1 Flexosel FlexNAcI   +
885.43                Flexose4
926.46                Flexose3 FlexNAcI           + + +
990.28                Neu5Ac1 Flexosel
                        FlexNAcI-Asn
967.43                Flexose2FlexNAc2
1053.62               Neu5Ac1 Flexosel
                        FlexNAcI Fud
1078.01               Flexose2 FlexNAc2-Asn
1089.53               Flexose5
1130.56               Flexose4 FlexNAcI           + + +
1171.58               Flexose3FlexNAc2
1212.58               Flexose2FlexNAc3
1240.61               Neu5Ac2 Flexosel FlexNAcI   +
1274.62               Flexose3FlexNAc1 Fuc3       +
1293.63               Flexose6
1334.66               Flexose5FlexNAc1            + +
1346.38               Fucosel Flexose3FlexNAc2
1375.68               Flexose4FlexNAc2
1416.65               Flexose3FlexNAc3
1439.43               Neu5Ac1 Flexose2Flex-
                        NAc2-Asn
1497.73               Flexose7
1532.76               Neu5Ac1 Hexose3HexNAc2      +/-
1538.76               Hexose6HexNAc1              + +
1550.58               Fucosel Hexose4HexNAc2
1578.66               Sial3HexoseHexNAc1
1614.72               Siall Hexosel HexNAc4
1620.81               Hexose 4HexNAc3
1661.76               Hexose3HexNAc4
1701.83               Hexose8
1736.86               Neu5Ac1 Hexose4HexNAc2      +/-
1742.86               Hexose7HexNAc1
1824.91               Hexose5HexNAc3
1875.90               Hexose8Fuc1
1881.71               Neu5Ac1 Flexose2FlexNAc2
1906.85               Flexose3FlexNAc5
1946.96               Flexose8FlexNAc1
1981.98               Neu5Ac1 Flexose4FlexNAc3    +/-
2151.06               Flexose9FlexNAc1
2173.21               Flexose5 FlexN Ac3 Fuc2
2186.08               Neu5Ac1 Flexose5FlexNAc3    +/-
2274.14               Flexose6FlexNAc4
2343.16               Neu5Ac2 Flexose4FlexNAc3
2547.26               Neu5Ac2 Hexose5 HexN Ac3    +/-
2629.16               Neu5Ac2 Hexose3 HexNAc5
2996.48               Neu5Ac2 Hexose6HexNAc4
3357.66               Neu5Ac3Hexose6HexNAc4

m/z       Predicted     Predicted components      [alpha]-Mannosidosis
          structure                               (n = 4)
             (a)

599.0                 Fucosel HexNAc1-Asn
629.1                 Hexosel HexNAcI-Asn
651.32                Hexose2Fuc1
681.33                Flexose3
692.35                Flexosel FlexNAcI Fud
722.36                Flexose2FlexNAc1            + + + +
825.86                Flexose2Fuc2
838.42                Neu5Ac1 Flexose2            +
879.43                Neu5Ac1 Flexosel FlexNAcI   +
885.43                Flexose4
926.46                Flexose3 FlexNAcI           + + +
990.28                Neu5Ac1 Flexosel
                        FlexNAcI-Asn
967.43                Flexose2FlexNAc2
1053.62               Neu5Ac1 Flexosel
                        FlexNAcI Fud
1078.01               Flexose2 FlexNAc2-Asn
1089.53               Flexose5
1130.56               Flexose4 FlexNAcI           + + +
1171.58               Flexose3FlexNAc2
1212.58               Flexose2FlexNAc3
1240.61               Neu5Ac2 Flexosel FlexNAcI   +
1274.62               Flexose3FlexNAc1 Fuc3       +
1293.63               Flexose6
1334.66               Flexose5FlexNAc1            + + +
1346.38               Fucosel Flexose3FlexNAc2
1375.68               Flexose4FlexNAc2
1416.65               Flexose3FlexNAc3
1439.43               Neu5Ac1 Flexose2Flex-
                        NAc2-Asn
1497.73               Flexose7
1532.76               Neu5Ac1 Hexose3HexNAc2
1538.76               Hexose6HexNAc1              + + +
1550.58               Fucosel Hexose4HexNAc2
1578.66               Sial3HexoseHexNAc1
1614.72               Siall Hexosel HexNAc4
1620.81               Hexose 4HexNAc3
1661.76               Hexose3HexNAc4
1701.83               Hexose8
1736.86               Neu5Ac1 Hexose4HexNAc2
1742.86               Hexose7HexNAc1              + + +/+ +
1824.91               Hexose5HexNAc3
1875.90               Hexose8Fuc1
1881.71               Neu5Ac1 Flexose2FlexNAc2
1906.85               Flexose3FlexNAc5
1946.96               Flexose8FlexNAc1            + + +/+ +
1981.98               Neu5Ac1 Flexose4FlexNAc3
2151.06               Flexose9FlexNAc1            + + +/+ +
2173.21               Flexose5 FlexN Ac3 Fuc2
2186.08               Neu5Ac1 Flexose5FlexNAc3
2274.14               Flexose6FlexNAc4
2343.16               Neu5Ac2 Flexose4FlexNAc3
2547.26               Neu5Ac2 Hexose5 HexN Ac3
2629.16               Neu5Ac2 Hexose3 HexNAc5
2996.48               Neu5Ac2 Hexose6HexNAc4
3357.66               Neu5Ac3Hexose6HexNAc4

m/z       Predicted     Predicted components        GM2       Pompe
          structure                               (n = 2)    disease
             (a)                                             (n = 11)

599.0                 Fucosel HexNAc1-Asn
629.1                 Hexosel HexNAcI-Asn
651.32                Hexose2Fuc1
681.33                Flexose3                               + + + +
692.35                Flexosel FlexNAcI Fud
722.36                Flexose2FlexNAc1
825.86                Flexose2Fuc2
838.42                Neu5Ac1 Flexose2            +          +
879.43                Neu5Ac1 Flexosel FlexNAcI   +          +
885.43                Flexose4                               + + + +
926.46                Flexose3 FlexNAcI
990.28                Neu5Ac1 Flexosel
                        FlexNAcI-Asn
967.43                Flexose2FlexNAc2            + + +
1053.62               Neu5Ac1 Flexosel
                        FlexNAcI Fud
1078.01               Flexose2 FlexNAc2-Asn
1089.53               Flexose5                               + + +
1130.56               Flexose4 FlexNAcI
1171.58               Flexose3FlexNAc2
1212.58               Flexose2FlexNAc3            + + +
1240.61               Neu5Ac2 Flexosel FlexNAcI   +          +
1274.62               Flexose3FlexNAc1 Fuc3       +          +
1293.63               Flexose6                               + + +/+ +
1334.66               Flexose5FlexNAc1
1346.38               Fucosel Flexose3FlexNAc2
1375.68               Flexose4FlexNAc2
1416.65               Flexose3FlexNAc3            + + +
1439.43               Neu5Ac1 Flexose2Flex-
                        NAc2-Asn
1497.73               Flexose7                               + + +/+ +
1532.76               Neu5Ac1 Hexose3HexNAc2
1538.76               Hexose6HexNAc1
1550.58               Fucosel Hexose4HexNAc2
1578.66               Sial3HexoseHexNAc1
1614.72               Siall Hexosel HexNAc4
1620.81               Hexose 4HexNAc3
1661.76               Hexose3HexNAc4              + +
1701.83               Hexose8                                + + +/+ +
1736.86               Neu5Ac1 Hexose4HexNAc2
1742.86               Hexose7HexNAc1
1824.91               Hexose5HexNAc3
1875.90               Hexose8Fuc1
1881.71               Neu5Ac1 Flexose2FlexNAc2
1906.85               Flexose3FlexNAc5            + +
1946.96               Flexose8FlexNAc1
1981.98               Neu5Ac1 Flexose4FlexNAc3
2151.06               Flexose9FlexNAc1
2173.21               Flexose5 FlexN Ac3 Fuc2
2186.08               Neu5Ac1 Flexose5FlexNAc3
2274.14               Flexose6FlexNAc4
2343.16               Neu5Ac2 Flexose4FlexNAc3
2547.26               Neu5Ac2 Hexose5 HexN Ac3
2629.16               Neu5Ac2 Hexose3 HexNAc5
2996.48               Neu5Ac2 Hexose6HexNAc4
3357.66               Neu5Ac3Hexose6HexNAc4

(a) Mannoze; galactose, glucose; N-acetylneuraminic acid; fucose;
GlcNac; N-acetylgalactosamine

(b) +, Relative abundance of FOS.

(c) *, Structure could not be predicted from common known FOS.
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Article Details
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Title Annotation:General Clinical Chemistry
Author:Xia, Baoyun; Asif, Ghazia; Arthur, Leonard; Pervaiz, Muhammad A.; Li, Xueli; Liu, Renpeng; Cummings,
Publication:Clinical Chemistry
Article Type:Report
Date:Sep 1, 2013
Words:6745
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