Glycosylation Analysis for Congenital Disorders of Glycosylation

UNIT 17.18

Xueli Li,1 Mohd A. Raihan,1 Francis Jeshira Reynoso,2 and Miao He1,3 1

Palmieri Metabolic Disease Laboratory, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 2 Department of Genetics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 3 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Congenital disorders of glycosylation (CDG) are a group of diseases with highly variable phenotypes and inconsistent clinical features. Since the first description of a CDG in 1980, approximately 100 disorders have been identified. Most of these are defects in protein glycosylation, although an increasing number are defects of glycolipid or proteoglycan biosynthesis. A group of biochemical markers has been used to characterize protein glycosylation abnormalities in CDG. This unit describes three protocols that can be used to measure plasma or serum carbohydrate deficient transferrin (CDT) profile, N-glycan profile, and O-glycan profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). The quantification of particular biomarkers, such as T antigens or sialylated T antigens, could also be achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These techniques can be used to identify a majority of patients with defects in protein glycosylation, although different techniques, such as flow cytometry with immunostaining, are necessary to detect defects in glycolipid or proteoC 2015 by John Wiley glycan biosynthesis which is not included in this unit.  & Sons, Inc. Keywords: transferrin r N-linked glycan r O-linked glycan r congenital disorders of glycosylation r MALDI-TOF-MS r LC-MS/MS r ThomsenFriedenreich antigen r N-acetyl-galactosamine

How to cite this article: Li, X., Raihan, M.A., Reynoso, F.J., and He, M. 2015. Glycosylation analysis for congenital disorders of glycosylation. Curr. Protoc. Hum. Genet. 86:17.18.1-17.18.22. doi: 10.1002/0471142905.hg1718s86

INTRODUCTION The glycosylation system is composed of more than 300 proteins such as glycosyltransferases, glycan-modifying glycosidases, and transporters located in different cellular compartments including the cytosol, endoplasmic reticulum, and Golgi apparatus. It is estimated that at least 2% of the human genome codes for proteins involved in this vital biochemical pathway. Congenital disorders of glycosylation (CDG) are an inherited group of disorders in which the synthesis or the structure of glycans is altered. The clinical presentation is heterogeneous and multisystemic. Common clinical manifestations include developmental delay, failure to thrive, microcephaly, coagulopathy, abnormal brain MRI including cerebral and/or cerebellar atrophy, cell migration abnormalities, and immune dysfunction. Most of these disorders follow autosomal recessive inheritance patterns, but X-linked, dominant, and de novo mutations have also been identified. The large majority of these are diseases of protein hypoglycosylation, but in recent years Current Protocols in Human Genetics 17.18.1-17.18.22, July 2015 Published online July 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142905.hg1718s86 C 2015 John Wiley & Sons, Inc. Copyright 

Biochemical Genetics

17.18.1 Supplement 86

Table 17.18.1 Analysis of Common of Type I, Type II, and Mixed Type I and II CDG Subtypes

Disorder

Plasma or serum CDT

Plasma or serum N-glycan

Plasma or serum O-glycan

PMM2-CDG

Abnormal

Abnormal

Normal

MPI-CDG

Abnormal

Abnormal

Normal

ALG1-CDG

Abnormal

Abnormal

Normal

ALG12-CDG

Abnormal

Abnormal

Normal

MAN1B1-CDG

Abnormal

Abnormal

Normal

DPAGT-CDG

Abnormal

Normal

Normal

ALG8-CDG

Abnormal

Normal

Normal

MOGS-CDG

Normal/abnormal

Abnormal

Normal

ATP6V0A2-CDG

Abnormal/normal

Abnormal

Abnormal

COG7-CDG

Abnormal

Abnormal

Abnormal

COG8-CDG

Abnormal

Abnormal

Abnormal

GNE-CDG

Normal

Normal

Abnormal

PGM3-CDG

Normal

Abnormal/normal

Abnormal

PGM1-CDG

Abnormal

Abnormal

Abnormal

several defects in lipid glycosylation have also been identified. Most protein glycosylation disorders are due to defects in the N-glycosylation pathway. The remaining ones affect the O-glycosylation pathway or combined N- and O-glycosylation pathways. Over 100 CDGs have been discovered to date, making discoveries in the field of glycobiology more relevant than ever for diagnostics and therapeutics (Freeze et al., 2014). Routine screening for protein glycosylation disorders includes: (1) carbohydrate deficient transferrin (CDT) analysis to detect protein hypoglycosylation, (2) N-glycan analysis to detect abnormal intermediate metabolites and structural changes in the N-linked glycosylation pathway, and (3) O-glycan analysis to detect disorders in combined N- and O-glycosylation disorders as well as isolated N-acetyl-D-galactosamine (GalNAc) type O-linked protein glycosylation disorders. All of the methods described utilize matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for analysis except for the quantification of the Thomsen-Friedenreich antigen (T antigen) and sialylated T (ST) antigen which uses liquid chromatography-tandem mass spectrometry (LC-MS/MS; Xia et al., 2013). Other methods, such as liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESI-MS), could also be used for CDT analysis (Lacey et al., 2001). The focus of the protocols described here is on the preparation of samples. Prior experience with mass spectrometry is required in order to perform these analyses. Application of these three analyses to common subtypes of CDG is shown in Table 17.18.1. BASIC PROTOCOL 1

Glycosylation Analysis for Congenital Disorders of Glycosylation

ANALYSIS OF CDT BY MALDI-TOF Transferrin is the second most abundant glycoprotein in human plasma after immunoglobulins. Each transferrin molecule has two N-linked glycosylation sites, both of which are utilized and ultimately occupied by typical N-linked biantennary glycans, with a molecular weight of the diglycosylated transferrin monomer at 79 kDa. Patients with the most common glycosylation disorders generally have monoglycosylated transferrin (loss of one glycan; molecular weight of 77 kDa) and sometimes non-glycosylated transferrin (aglycosylated molecules that lack both N-glycans; molecular weight of 75 kDa). These

17.18.2 Supplement 86

Current Protocols in Human Genetics

hypoglycosylated transferrin species are known as CDT. This CDT test is designed to measure the presence of abnormally glycosylated transferrin to identify patients with type I CDG. Transferrin in the plasma or serum is enriched by using an anti-human transferrin antibody-coupling affinity column following the depletion of protein albumin and immunoglobulins before it is analyzed by mass spectrometry. Affinity spin columns to deplete albumin and immunoglobulins are commercially available, and affinity spin columns to bind transferrin are made by conjugating anti-transferrin polyclonal antibodies (rabbit anti-human or goat anti-human) to affinity resin (see Support Protocol 2). CDT by MALDI-TOF analysis provides higher sensitivity than most other methods with detection limits of mono- and diglycosylated transferrin as low as 0.01. However, CDT by MALDI-TOF does not have the resolution to detect type II CDG and should be offered in combination with N-glycan profiling for a comprehensive screening for a majority of N-linked CDGs.

Materials Normal control plasma (see recipe) Positive control plasma (see recipe) Patient samples (i.e., plasma or serum in tubes containing heparin or EDTA) ProteoSeek Antibody-Based Albumin/IgG Removal Kit (e.g., Pierce) Polyclonal rabbit anti-human transferrin antibody-coupling affinity column (see Support Protocol 2) Washing solution (resin storage buffer; see recipe) Elution buffer (see recipe) Protein determination reagent (for BCA assay; e.g., Bio-Rad) MALDI matrix solution (α-cyano-4-hydroxycinnamic acid; see recipe) Benchtop shaker Centrifuge YM-10 Microcon filter (e.g., Millipore) 2-ml microcentrifuge tubes Lyophilizer MALDI plate (384 well; e.g., ABSciex) MALDI-TOF mass spectrometer (e.g., ABSciex 4800 plus) Computer running spreadsheet program (e.g., Microsoft Excel) Set up controls and samples 1. Set up a number of 1.5-ml microcentrifuge tubes for normal control (#1), positive control (#2), and patient samples (#3 and up). All plasma or serum specimens should be kept on ice after being thawed.

Deplete the albumin and IgG from plasma/serum 2. Label albumin/IgG antibody-coupling gel columns (from ProteoSeek Kit) the same as samples: normal control (#1), positive control (# 2), and patient samples (# 3 and up). 3. Remove cap of albumin/IgG antibody-coupling gel column. Add 40 μl plasma or serum for each sample (including controls and patient samples) into albumin/IgG antibody-coupling gel column, and then cap column. 4. Shake column gently by hand two to three times. 5. Shake column on a benchtop shaker at 300 rpm for 30 min at room temperature.

Biochemical Genetics

17.18.3 Current Protocols in Human Genetics

Supplement 86

During the 30-min shaking, mix the gel gently by hand two to three times.

6. Twist off bottom closure of gel column and loosen cap. 7. Place column into a new 1.5-ml microcentrifuge tube and centrifuge 2 min at 1000 × g, room temperature. 8. Save the flowthrough from the columns for later use. The depletion column can be kept at 4°C until column regeneration (see Support Protocol 1).

Purification of transferrin by affinity column 9. Remove cap of transferrin affinity column, load the flowthrough from the albumin/IgG antibody-coupling column to the transferrin affinity column, and cap the transferrin affinity column. 10. Shake transferrin affinity column at 300 rpm for 30 min at room temperature on a benchtop shaker. During the 30 min shaking, mixing the gel gently by hand two to three times is recommended.

11. Twist off bottom closure and loosen cap of transferrin affinity column. Place column into a new 1.5-ml microcentrifuge tube and centrifuge 2 min at 500 × g, room temperature. 12. Add 200 μl washing solution to transferrin affinity column, and centrifuge 1 min at 500 × g, room temperature; discard the flowthrough. Repeat this step four times for a total of five washes. 13. Add 200 μl elution buffer, and incubate column at room temperature for 2 min. Then, centrifuge 1 min at 500 × g, room temperature. Repeat this step once, and collect the combined flowthrough in a new 1.5-ml microcentrifuge tube. Keep the transferrin affinity column at 4°C until column regeneration (see Support Protocol 1).

Desalt the purified transferrin 14. To prewash YM-10 Microcon filter (mini), place YM-10 Microcon filter in a 2-ml microcentrifuge tube, add 500 μl water, and centrifuge 15 min at 10,000 × g, room temperature. Repeat this step one more time. 15. Load the eluate from the transferrin affinity column to prewashed YM-10 Microcon filter, centrifuge 15 min at 10,000 × g, room temperature, and discard the flowthrough. 16. Add 300 μl water, centrifuge 15 min at 10,000 × g, room temperature, and discard the flowthrough. Perform this step a total of three times. 17. Turn the YM-10 Microcon filter upside down in a new 1.5-ml microcentrifuge tube, and centrifuge 2 min at 6000 × g, room temperature, to collect the desalted transferrin protein. 18. Add 250 μl water to the tube, and wash the filter by pipetting the solution up and down four times. Glycosylation Analysis for Congenital Disorders of Glycosylation

19. Combine solutions from step 17 and step 18 and lyophilize it overnight. 20. Add 40 μl water to resuspend transferrin protein. Use 10 μl of resuspended protein for BCA protein assay.

17.18.4 Supplement 86

Current Protocols in Human Genetics

A

B 100

26316

100

80 Relative intensity %

Relative intensity %

80

26282

60

40

20

60

40 25648 20

25704

24983

0

0 23877

30480

27178 m/z

C

23813

39294

100

27544 m/z

31275

+1

+2

78399

Relative intensity %

80

60

+3 38428

40

20

0 9979

+4

37444

38176

76670

74867

66373

94570

122767

150965

m/z

Figure 17.18.1 Typical normal and abnormal plasma transferrin profiles by MALDI-TOF. (A) The transferrin profile from a normal control by MALDI-TOF analysis. (B) The transferrin profile from a patient with type I CDG. (C) The complete MALDI-TOF spectra of type I CDG transferrin with different charge statuses. The third charged spectrum is used to calculate the ratio between mono-/diglycosylated and a-/diglycosylated transferrin.

21. Dilute samples to 0.25 mg/ml using Milli-Q water.

Spot samples on the MALDI plate 22. Spot 0.5 μl transferrin solution and 0.5 μl α-cyano-4-hydroxycinnamic acid matrix solution on the MALDI plate. Record sample ID and its location on the MALDI plate.

23. Place the MALDI plate on a clean benchtop at room temperature until samples are dried.

Biochemical Genetics

17.18.5 Current Protocols in Human Genetics

Supplement 86

Mono/Diglycosylated transferrin intensity ratios

A y=0.964x+0.013

1.0

R2=1.00

0.8 0.6 0.4 0.2 0.0 0.0

0.2

0.4

0.6

0.8

1.0

Mono/Diglycosylated transferrin molar ratios

B

LC-ESI-MS ratio

1.0

0.8

0.6

0.4

y=0.98x+0.036 R2=0.98

0.2

0.0

0.0

0.2

0.4

0.6

0.8

1.0

MALDI-TOF ratio Figure 17.18.2 Accuracy of CDT analysis by MALDI-TOF. (A) The association between the measured and molar ratio of mono- and diglycosylated transferrins. (B) The comparison study of CDT measurement between MALDI-TOF and LC-ESI-MS.

Scan samples on MALDI-TOF Transferrin analysis was carried out on a MALDI-TOF/TOF 4800 Analyzer (ABSciex). The instrument was operated in positive linear mode, and laser power was set at 4880 (arbitrary units), digitizer 0.82 (arbitrary units). The processing method was the default positive linear method. The 4000 series explorer software (ABSciex) was used to acquire, analyze, and export the data into Microsoft Office files. The average of three scans was acquired as the final chromatogram. The chromatogram was exported to Microsoft PowerPoint. An example of a transferrin chromatogram is shown in Figure 17.18.1A.

Glycosylation Analysis for Congenital Disorders of Glycosylation

Calculating the ratio of monotransferrin over ditransferrin On the spreadsheet exported from the MALDI, look for the 3+ m/z charge of intensity (peak height). The intensity of 3+ transferrin is used to calculate the ratio between monoand diglycosylated transferrin and a- and diglycosylated transferrin. The diglycosylated transferrin 3+ m/z is at 26518. The monoglycosylated transferrin 3+ m/z is at 25784.

17.18.6 Supplement 86

Current Protocols in Human Genetics

The aglycosylated transferrin 3+ m/z is at 25049.3. A cutoff of 0.1 is used for the mono-/diglycosylated transferrin ratio, while a cutoff of 0.03 is used for the a/diglycosylated transferrin ratio (Lacey et al., 2001). Aglycosylated transferrin species are usually absent in the majority of the samples from normal controls. Transferrin with different charge states could be detected by MALDI-TOF using this method (Fig. 17.18.1AC). As the charge state increases, the resolution of each transferrin glycoform increases, while the intensity of the mass spectrometry signal decreases. We found at 3+ charge status, aglycosylated and monoglycosylated transferrin separate well from diglycosylated species, and the intensity of the mass spectrometry signal is proportional to the molar ratio of transferrin glycoforms. The intensity is high enough to allow the detection limit of the mono-/ditransferrin ratio to be as low as 0.01 (Fig. 17.18.2A). The association between the measured mono-/ditransferrin ratio and the molar ratio of mono/ditransferrin is shown in Figure 17.18.2A. A comparison study between MALDI-TOF and LC-ESI-MS is shown in Figure 17.18.2B. The observed m/z difference between diglycosylated transferrin and monoglycosylated transferrin should be between 634 and 734 Da. There are variations in the molecular mass of transferrin in the normal population due to the presence of benign variants in the transferrin gene. However, the molecular weight between each glycoforms should remain the same in the normal population. The observed m/z of diglycosylated transferrin 3+ could vary between 26,200 and 26,570; monoglycosylated transferrin 3+, between 25,450 and 25,830; and aglycosylated transferrin 3+, between 24,900 and 25,080.

REGENERATION OF COLUMNS The albumin/IgG antibody-coupling gel column can be regenerated together with the anti-human transferrin affinity column. Regenerating the columns after each batch will help maintain column performance for at least 2 years and also prevent contamination from carryover. The yield and purity of transferrin should be used to monitor column performance.

SUPPORT PROTOCOL 1

Materials Albumin/IgG antibody-coupling gel column (from Basic Protocol 1) Transferrin affinity column (from Basic Protocol 1) Washing solution (see recipe) Elution buffer (see recipe) Centrifuge 1. Add 400 μl washing solution to column, and centrifuge 2 min at 800 × g, room temperature. Perform this step six times, discarding the flowthrough. A vacuum apparatus can be used instead of centrifugation, which allows for washing and regenerating multiple columns at the same time.

2. Add 200 μl elution buffer to column, incubate at room temperature for 2 min, and then centrifuge 2 min at 800 × g, room temperature. Perform this step ten times, discarding the flowthrough. 3. Add 400 μl washing solution, and centrifuge 2 min at 800 × g, room temperature. Perform this step four times, discarding the flowthrough. 4. Cap the bottom of column, add 200 μl washing solution, and then cap the top of column. Store the column at 4°C. Biochemical Genetics

17.18.7 Current Protocols in Human Genetics

Supplement 86

SUPPORT PROTOCOL 2

PREPARATION OF THE ANTI-TRANSFERRIN AFFINITY COLUMN Anti-transferrin affinity columns are not readily available commercially and often need to be custom made. This protocol describes a method to make anti-transferrin resin, which could be used to make mini spin columns (as described here) or a liquid chromatography (LC) column for the LC-ESI-MS based method (Lacey et al., 2001).

Materials Polyclonal rabbit anti-human transferrin antibody (e.g., Dako or Bio-Rad) 10 mM MOPS-NaOH buffer (pH 7.5; see recipe) Affi-Gel 10 resin (e.g., Bio-Rad) 0.1 M MOPS-NaOH buffer (pH 7.5; see recipe) 1 M ethanolamine-HCl buffer (pH 8.0, see recipe) Washing buffer (see recipe) Slide-A-Lyzer 10K dialysis cassettes (e.g., Pierce) 5-ml syringe 15-ml plastic centrifuge tubes (e.g., Corning) Lyophilizer Nalgene Rapid-Flow filter unit, alpha PES membrane, 0.2 μm (e.g., Thermo Scientific) Benchtop shaker Centrifuge Empty mini spin column 1. To desalt anti-human transferrin antibody, transfer 2 ml polyclonal rabbit anti-human transferrin antibody into Slide-A-Lyzer 10K Dialysis Cassettes using a 5-ml syringe, and dialyze the antibody overnight against 1 liter of 10 mM MOPS-NaOH buffer (pH 7.5). 2. Aspirate the desalted antibody from dialysis cassettes with a syringe, and transfer into a 15-ml plastic centrifuge tube. Dry the anti-transferrin antibody solution with a lyophilizer, and store the resulting powder at 4°C. 3. To wash Affi-Gel 10 resin, move 12 ml Affi-Gel 10 resin slurry into a Nalgene Rapid-Flow filter unit connected by a vacuum pump, and aspirate all liquid. Then, wash Affi-Gel 10 resin with 200 ml cooled water, and transfer resin into a 15-ml plastic centrifuge tube. More cooled water can be used until the resin is washed thoroughly and free of organic solvent. This step can be performed in a chemical hood if necessary.

4. To couple transferrin antibody with Affi-Gel 10 resin, dissolve the purified polyclonal rabbit anti-human transferrin antibody (step 2) in 4 ml of 0.1 M MOPS-NaOH buffer (pH 7.5). Transfer antibody solution into the washed resin (step 3). Shake the mixture 1 hr at room temperature. 5. Add 0.6 ml of 1 M ethanolamine-HCl buffer (pH 8.0), and shake 1 hr at room temperature. 6. Centrifuge 1 min at 800 × g, 4 °C. 7. Discard supernatant, and add 6 ml washing buffer. Centrifuge 1 min at 800 × g, 4 °C. Glycosylation Analysis for Congenital Disorders of Glycosylation

8. Repeat step 7, then store the resin coupling the anti-transferrin antibody in 12 ml washing buffer at 4°C. Resin is stable for up to 2 years.

17.18.8 Supplement 86

Current Protocols in Human Genetics

9. To pack transferrin affinity column, mix the slurry of Affi-Gel 10 resin coupling the anti-transferrin antibody gently, and pipette 160 μl of slurry into a small, empty mini spin column. Centrifuge 1 min at 800 × g, room temperature, to pack the transferrin affinity column (or adjust the slurry volume to ensure the precipitated resin volume is about 100 μl). 10. Twist on the bottom of column, add 200 μl washing buffer, and cap the top of column. Store at 4°C.

PREPARATION OF MONOGLYCOSYLATED TRANSFERRIN PROTEIN Monoglycosylated transferrin protein can be made from pure human transferrin protein using peptide-N-glycosidase F (PNGase F) digestion. This method will produce >90% pure monoglycosylated transferrin protein that can be used to spike into normal plasma to make quality control material.

SUPPORT PROTOCOL 3

Materials Pure transferrin protein (e.g., Sigma-Aldrich) PNGase F enzyme and digestion buffer (e.g., New England Biolabs) Anti-human transferrin affinity column (see Basic Protocol 1 and Support Protocol 2) Protein assay kit YM-10 Microcon filter (e.g., Millipore) MALDI plate (384 well; e.g., ABSciex) MALDI-TOF mass spectrometer (e.g., ABSciex 4800 plus) 1. Dissolve 1 mg pure transferrin protein in 100 μl water (10 mg/ml). Add 10 μl digestion buffer and 1 μl PNGase F (250,000 units/ml). Incubate in a water bath at 37°C for 2 hr. 2. Purify the digested mixture through an anti-transferrin affinity column as described in Basic Protocol 1. 3. Desalt the monoglycosylated transferrin using a YM-10 Microcon filter as described in Basic Protocol 1. 4. Determine the protein concentration using a protein assay kit. 5. Validate the purity of monoglycosylated transferrin using MALDI-TOF or LC-ESIMS analysis. 6. Aliquot and store at −80°C. Aliquots are stable for 2 years.

PLASMA/SERUM N-GLYCAN PROFILE ANALYSIS Although transferrin analysis reliably identifies the majority of the N-linked hypoglycosylation disorders, it does not pinpoint any specific CDG subtypes. It has been estimated that 30% of patients with protein hypoglycosylation lack a diagnosis of a specific CDG subtype. Plasma/serum N-glycan profile analysis is currently the only clinical test that could provide a screen for a number of CDG subtypes including PMM2CDG (CDG-Ia), MPI-CDG (CDG-Ib), ALG1-CDG (CDG-Ik), ALG12-CDG (CDG-Ig), Man1B1-CDG, MOGS-CDG (CDG-IIb), B4GALT1-CDG (CDG-IId), MGAT2-CDG (CDG-IIa), SLC35A1-CDG (CDG-IIf), SLC35A2-CDG, PGM1-CDG, PGM3-CDG,

BASIC PROTOCOL 2

Biochemical Genetics

17.18.9 Current Protocols in Human Genetics

Supplement 86

TMEM165-CDG, Cutis Laxa type IIA (ATP6V0A2-CDG) as well as conserved oligomeric Golgi complex (COG) deficiencies (Xia et al., 2013). The number of CDG subtypes with specific structural alterations continues to grow as more genes are found to be responsible for glycosylation deficiencies or abnormalities; and more new CDG subtypes have been recognized by N-glycan profile in recent years. Although some Golgiassociated glycosylation defects (previously known as type II CDG) can be detected by transferrin analysis using isofocusing electrophoresis or ESI-MS methods, plasma/serum N-glycan profiling is a more sensitive and specific tool for this group of CDGs. N-glycan profiles are obtained by first releasing N-linked glycans from total glycoproteins in plasma or serum using PNGase F digestion—a glycanase that specifically cleaves Nlinked glycans from glycoproteins. The released N-glycans are then purified through a C18 column followed by a carbograph column. Next, they are permethylated by a traditional liquid-liquid permethylation method and desalted by organic extraction with chloroform and water. Finally, permethylated N-glycan sodium adducts are analyzed by MALDI-TOF-MS.

Materials Plasma or serum samples, including normal control and positive control (see recipe) 10 μM mannopentose (internal standard) PNGase F enzyme with associated digestion buffers (e.g., New England Biolabs) Sep-Pak C18 3 cc Vac cartridges (e.g., Waters) Methanol Extract-Clean SPE Carbograph column (e.g., UCT) 80% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid (TFA) in water (Optima LC/MS; e.g., Fischer Scientific) 30% (v/v) acetonitrile with 0.1% (v/v) TFA in water (Optima LC/MS; e.g., Fischer Scientific) Nitrogen source Anhydrous dimethyl sulfoxide (DMSO) NaOH beads (20-40 mesh) Iodomethane Chloroform MALDI matrix solution (2,5-dihydroxybenzoic acid, DHB; see recipe) Water bath 15-ml plastic centrifuge tubes (e.g., Corning) Centrifuge Lyophilizer 5-ml syringe and needle Benchtop shaker 2-ml microcentrifuge tube MALDI-TOF plate (384 well; e.g., ABSciex) MALDI-TOF mass spectrometer (e.g., ABSciex 4800 plus) Release N-glycans from total glycoproteins in plasma or serum 1. Set up 1.5-ml microcentrifuge tubes for normal control (#1), positive control (#2), and samples (#3 and up). 2. Pipet 20 μl of plasma or serum into 1.5-ml microcentrifuge tube. Glycosylation Analysis for Congenital Disorders of Glycosylation

3. Add 140 μl Milli-Q water, 20 μl of 10 μM mannopentose (internal standard), and 20 μl denature buffer (from PNGase F enzyme kit) to plasma or serum samples so the total volume of the mixture is 200 μl. Vortex mixture briefly.

17.18.10 Supplement 86

Current Protocols in Human Genetics

4. Incubate mixture in a water bath at 100°C for 10 min. 5. Cool mixture to room temperature by placing tubes on the benchtop for 5 to 10 min. 6. Add 25 μl NP-40, 25 μl of 10X G7 Reaction Buffer, and 1 μl PNGase F (all from PNGase F enzyme kit). 7. Mix reaction well by pipetting up and down a few times. Incubate tube at 37°C for 16 hr or overnight. 8. Heat tube in a 100°C water bath for 10 min to stop the PNGase F digestion, and then label one set of 15-ml plastic centrifuge tubes. 9. Centrifuge the digested sample 4.5 min at 10,000 × g, room temperature. 10. Carefully transfer supernatant into a labeled 15-ml plastic centrifuge tube. Add Milli-Q water to dilute the mixture to total volume of 8 ml. 11. Activate Sep-Pak C18 column by adding 3 ml methanol to column. 12. Wash Sep-Pak C18 by adding 12 ml (4 full column volume) Milli-Q water. 13. Label and activate carbograph column by adding 3.0 ml of 80% (v/v) acetonitrile with 0.1% (v/v) TFA to column. 14. Wash carbograph column by adding 12 ml (4 full column volumes) of Milli-Q water. 15. Stack Sep-Pak C18 column on top of carbograph column so that eluted sample from Sep-Pak C18 column will flow into carbograph column. 16. Load samples into the Sep-Pak C18 columns. Then, add 1 ml Milli-Q water to all sample tubes, and reload it into Sep-Pak C18 column. Repeat this one more time. Discard all flowthrough. 17. Remove Sep-Pak C18 column, and then wash carbograph column with 12 ml (4 full column volume) Milli-Q water. 18. Label a set of 15-ml plastic centrifuge tubes as collection tubes. Then, match and place labeled carbograph column within the collection tubes. 19. Elute the glycans from carbograph column with 4.5 ml of 30% (v/v) acetonitrile with 0.1% (v/v) TFA. 20. Remove the acetonitrile in the solution under nitrogen in a chemical hood for 0.5 to 1 hr. The remaining volume should be about 3.0 to 3.2 ml. The elution with acetonitrile cannot be dried with a lyophilizer because the pump would be damaged by acetonitrile.

21. Freeze the remaining volume (about 0.5 to 1 hr) and lyophilize samples overnight.

Permethylation of N-glycans 22. Transfer 10 ml (>500 μl for each sample) anhydrous DMSO from original bottle by 5-ml syringe to a small glass bottle. Then, add about 400 mg NaOH beads to DMSO to make fresh NaOH-saturated DMSO slurry for each batch of permethylation reactions. 23. Add 100 μl DMSO to dried glycans in a 15-ml plastic centrifuge tube. 24. Vortex and spin briefly.

Biochemical Genetics

17.18.11 Current Protocols in Human Genetics

Supplement 86

0

1000

2000

3000

4000

0 1000

2792.4

Abnormal COG7-CDG

2000

3602.8 3776.9

3241.6

2966.5

2605.3

2227.1 1865.9

1375.8

3602.8

3776.9

3241.6

2966.5

2431.2 2605.3

2227.1

1783.9 1982.0

1375.8

IS

50

1579.8

%Intensity

Normal

50

1579.8

%Intensity

2431.2

B 100

2792.4

100

1661.8

A

3000

4000

Figure 17.18.3 Typical normal and abnormal plasma N-glycan profiles by MALDI-TOF. (A) N-glycan profile from a normal control. (B) N-glycan profile from COG7-CDG plasma. All the truncated glycans, along with Man5 GlcNAc2 and Man6 GlcNAc2 are increased in COG7-CDG plasma, while normal triantennary glycans are reduced.

25. Add 10 μl Milli-Q water to all samples. 26. Add 300 μl NaOH-saturated DMSO slurry to each 15-ml plastic centrifuge tube containing resuspended glycan samples. 27. Add 100 μl iodomethane to the reaction mixture. 28. Shake reaction mixture vigorously for 60 min on a benchtop shaker. 29. Centrifuge mixture 15 sec at 2000 × g, room temperature. 30. Carefully transfer supernatant into a new set of labeled 2-ml microcentrifuge tubes. 31. Add 800 μl Milli-Q water to each sample, and vortex immediately after addition. Then, add 600 μl chloroform to each sample, and vortex immediately after addition. 32. Spin microcentrifuge tube 1 min at 3000 × g, room temperature. 33. Remove the top layer, and add 1 ml Milli-Q water to the chloroform layer at the bottom. Then, vortex vigorously, and spin 1 min at 3000 × g, room temperature. 34. Repeat step 33 three more times. 35. Discard the water layer after the final wash, and dry the chloroform phase under the nitrogen in a chemical hood for 30 min.

Acquire N-glycan profile by MALDI-TOF/TOF 36. Add 20 μl of 50% methanol to the bottom of dried tube and vortex. 37. Spin samples down by centrifuging 1 min at 3000 × g, room temperature. 38. Spot 0.5 μl sample onto MALDI plate with 0.5 μl DHB matrix. If there is poor crystal formation after spotting, then vary the amount of sample and matrix. Glycosylation Analysis for Congenital Disorders of Glycosylation

Write down sample’s name or number and location before spotting.

39. Adjust laser intensity of mass spectrometer so the total intensity is at 1.0E + 4 or higher.

17.18.12 Supplement 86

Current Protocols in Human Genetics

Figure 17.18.4

N-glycan changes in type II CDG patients.

40. Acquire the final chromatogram as the average of three scans. Export the chromatogram to Microsoft PowerPoint. Examples of chromatograms are shown in Figure 17.18.3. 41. Export the peaks to Microsoft Excel. The area of the centroid is used to calculate the percent total abundance of each glycan peak as shown in Figure 17.18.4.

PLASMA/SERUM O-GLYCAN QUALITATIVE PROFILING BY MALDI-TOF-MS AND O-GLYCAN QUANTIFICATION BY LC-MS/MS GalNAc O-glycan is the major type of O-linked protein glycosylation in humans, which occurs exclusively in the Golgi apparatus. Although only one genetic disorder has been identified in the GalNAc O-glycan biosynthesis pathway (GALNT3-CDG; hyperphosphatemic familial tumoral calcinosis), GalNAc O-glycan profiling and quantification has proven to be a sensitive screening tool to detect Golgi-associated glycosylation disorders. This disorder comprises the majority of type II CDG with combined N- and O-linked glycosylation defects (Wopereis et al., 2007). In addition, the KM of CMP-sialic acid for GalNAc O-glycan specific sialyltransferase is much higher than the KM of CMPsialic acid for N-glycan specific sialyltransferase. Therefore, O-glycan quantification could also be used to screen for a functional deficiency of CMP-sialic acid, such as with the deficiency in GNE myopathy patients (Leoyklang et al., 2014). In human plasma or serum the majority of O-glycans are GalNAc O-glycans, while in muscle and heart

BASIC PROTOCOL 3

Biochemical Genetics

17.18.13 Current Protocols in Human Genetics

Supplement 86

tissue O-mannosyl glycans are abundantly present in addition to GalNAc O-glycans. The O-glycan method described here applies mainly to GalNAc O-glycans when it is used for plasma or serum O-glycan analysis. However, it can also be used for O-mannosylated glycans if O-glycans are obtained from muscle. The O-glycan profile in plasma or serum is qualitatively analyzed by MALDI-TOF-MS and quantified by LC-MS/MS. O-glycans in plasma or serum are first released by the traditional β-elimination method. The released carbohydrates are desalted with AG 50W-X8 resin and permethylated using solid phase permethylation. The permethylated O-glycans are initially analyzed by MALDI-TOF, and then T and ST antigen levels are quantified by LC-MS/MS. The trisaccharide raffinose is used as an internal standard. Purified T and ST antigen standards are used to generate calibration curves to calculate concentrations of plasma or serum T and ST antigens. In positive ESI-MS mode, permethylated T and ST antigens are well-ionized and detected (Xia et al., 2013).

Materials Plasma or serum samples, including normal control and positive control (see recipe) 50 μM galacto-N-biose (T antigen) 500 μM 3 -N-acetylneuraminyl-N-acetyllactosamine sodium salt (ST antigen) 10 μM D-(+)-raffinose pentahydrate (internal standard) 2 M sodium borohydrate in 0.1 M sodium hydroxide solution (NaBH4 -NaOH; make fresh; see recipe) 0.25 M acetic acid in methanol (see recipe) Nitrogen source Methanol AG 50W-X8 (see recipe; e.g., Bio-Rad) Anhydrous DMSO NaOH beads Iodomethane Chloroform MALDI matrix solution (DHB; see recipe) 15-ml plastic centrifuge tube (e.g., Corning) Oven 9-inch disposable Pasteur pipets Glass beads Lyophilizer 5-ml syringe and needle Empty ultra microspin column Centrifuge MALDI plate (384 well; e.g., ABSciex) MALDI-TOF mass spectrometer (e.g., ABSciex 4800 plus) High-performance liquid chromatography (HPLC) vial LC-MS/MS system (e.g., ABSciex 5500 system) Release O-glycans from serum protein (β-elimination) 1. Set up 15-ml plastic centrifuge tubes for standard curve (#1-6), normal control (#7), positive control (#8), and patient samples (#9 and up). All plasma or serum specimens should be kept on ice after they are thawed. Glycosylation Analysis for Congenital Disorders of Glycosylation

2. Prepare standard curve according to Table 17.18.2. Standards are prepared with 50 μM T antigen and 50 μM ST antigen working solutions.

17.18.14 Supplement 86

Current Protocols in Human Genetics

Table 17.18.2 Standard Curve for O-Glycan Profile

T antigen 50 μM T Standard concentration antigen curve # (μM) (μl)

ST antigen 50 μM ST Internal concentration antigen standard (μM) (μl) (μl)

Water (μl)

Total (μl)

1

50

1

500

1

25

73

100

2

100

2

1000

2

25

71

100

3

150

3

1500

3

25

69

100

4

200

4

2000

4

25

67

100

5

250

5

2500

5

25

65

100

6

500

10

5000

10

25

55

100

3. Add 80 μl water and 10 μl internal standard to 15-ml plastic centrifuge tubes for all controls and patient samples (except standards). 4. Add 10 μl plasma or serum for all control and patient tubes. 5. Prepare NaBH4 -NaOH solution. NaBH4 -NaOH must be made fresh and should be added into samples immediately once it is prepared. The NaBH4 bottle should be desiccated and is stable for one month once opened.

6. Add 100 μl NaBH4 -NaOH into all tubes (including standards) and vortex. 7. Incubate tubes in a 45°C oven for 16 hr (overnight). 8. Cool tubes to room temperature.

Purification of released O-glycans 9. Add 3 ml of 0.25 M acetic acid-methanol solution drop by drop to β-elimination mixture in a chemical hood to neutralize the reaction. Then, dry the mixture under a stream of nitrogen. Repeat this step one more time. A lot of air bubbles will be generated when 3 ml of 0.25 M acetic acid-methanol solution is added to β-elimination mixture.

10. Add 3 ml methanol, and dry under a stream of nitrogen. A small amount of white crystal will be visible after sample is dried

11. Add 2.0 ml water and vortex to dissolve the O-glycan. 12. Set up 9-inch disposable Pasteur pipets for all standards, controls, and patient samples. Put a glass bead in Pasteur pipet to turn it into an empty column for AG50W-X8 resin. 13. Pack AG 50W-X8 resin into Pasteur pipet. The height of the resin is naturally about 2 cm after precipitation.

14. Wash resin column two times by adding a full column volume of water. 15. Label a new set of 15-ml plastic centrifuge tubes, and put the new tubes under the packed resin columns for collecting flowthrough. 16. Load O-glycan samples (step 11) into the balanced columns, and collect the flowthrough.

Biochemical Genetics

17.18.15 Current Protocols in Human Genetics

Supplement 86

17. Rinse the previous sample tubes with 1 ml water, reload columns, and collect the flowthrough in the same tube. Repeat this step once. Total volume is 4 ml. 18. Freeze on dry ice and lyophilize overnight.

Permethylation of O-glycans 19. Pack NaOH column. a. Transfer 10 ml (>500 μl for each sample) anhydrous DMSO from reagent bottle by 5-ml syringe to a small bottle, and then add 400 mg NaOH beads in DMSO to make NaOH-saturated DMSO. b. Transfer >300 μl for each sample of anhydrous DMSO from reagent bottle by 5-ml syringe to an additional small bottle. c. Set up empty ultra microspin column on labeled 2-ml microcentrifuge tube for collection. d. Pack column with presoaked NaOH beads in DMSO (about 70% to 80% column volume). Cut the tapered ends of 1-ml pipet tips with a blade to increase the opening of the tip and facilitate pipetting the slurry of NaOH-saturated DMSO when packing NaOH column. Transfer 500 μl of the upper DMSO layer into an empty ultra microspin column first. Then transfer slurry.

e. Centrifuge the packed column 1 min at 1000 × g, room temperature, to remove excess DMSO. f. Add 200 μl DMSO in the column, and centrifuge 1 min at 1000 × g, room temperature. Cap both sides of the column. 20. Permethylate the glycan samples. a. Add 70 μl DMSO into dried sample (desalted and lyophilized glycans from step 18), and vortex. b. Add 1 μl water and 17.5 μl iodomethane to sample, and mix thoroughly. c. Uncap the top of the prepacked NaOH-saturated DMSO column, and load the glycan sample solution. d. Replace cap, and allow sample to migrate slowly through the column for 30 to 40 min at room temperature. e. Uncap both sides of column, and centrifuge 10 min at the lowest speed (500 to 800 × g), room temperature. f. Centrifuge 1 min at 8000 × g, room temperature, and save the eluent. 21. Desalt O-glycans by organic extraction. a. Add 200 to 300 μl water and 650 μl chloroform into the eluent, and vortex. b. Centrifuge 1 min at 3000 × g, room temperature, and aspirate the upper water layer with a pipet. c. Add 200 to 300 μl water, vortex, and centrifuge 1 min at 3000 × g, room temperature. Aspirate the upper water layer. Perform this step four times. d. Dry the chloroform phase with a stream of nitrogen. e. Add 20 μl of 50% methanol. Vortex well to fully resuspend the O-glycans before spotting on MALDI plate.

Glycosylation Analysis for Congenital Disorders of Glycosylation

Spot samples on the MALDI plate 22. Spot the 0.5 μl DHB matrix solution and 0.5 μl sample on a spot of the MALDI plate. Write down sample’s name or number and location before spotting.

23. Keep the plate at room temperature for 10 to 20 min until samples are dried.

17.18.16 Supplement 86

Current Protocols in Human Genetics

895.6

A 100

Normal

60

0 512.0

1311.8

895.6

100

80

1578.4

1845.0

Abnormal

681.4

I.S.

60 534.3 0 497.0

765.6

1034.2

1705.8

1344.9

20

1256.8

40

925.6

% Intensity

1045.2

793.5

B

778.6

1344.9

534.3

925.6

20

1705.8

40

1256.8

793.5

I.S. 681.4

% Intensity

80

1302.8

1571.4

1840.0

Figure 17.18.5 Typical plasma normal and abnormal O-glycan profiles by MALDI-TOF. (A) Oglycan profile from a normal control. (B) O-glycan profile from a GNE myopathy patient. T antigens are increased while the rest of the O-glycan species are reduced in the patient.

24. Scan samples on MALDI-TOF mass spectrometer with mass (m/z) range set to be from 500 to 4000. Adjust laser intensity so the total intensity is at 1.0E + 4 or higher. 25. Acquire the final chromatogram as the average of three scans. Export chromatogram to Microsoft PowerPoint. Chromatogram examples of plasma O-glycans are shown in Figure 17.18.5.

Quantification of O-glycans by LC-MS/MS 26. Add 480 μl of 25% methanol into the remaining sample and vortex (25× dilution). For LC-MS/MS injection, the sample dilution factor should be adjusted according to the sensitivity of LC-MS/MS system.

27. Transfer the diluted sample to an HPLC sample vial. Sample is ready for injection.

Biochemical Genetics

17.18.17 Current Protocols in Human Genetics

Supplement 86

Table 17.18.3 Quantification of Plasma or Serum O-glycans in Patients with Type II CDG and Normal Controls

O-glycan

T antigen (μM)

ST antigen (μM)

T/ST ratio

Control

1.03

13

0.058

TMEM165

1.67

9.45

0.177

PGM1 (mixed type I and II)

1.44

9.38

0.154

PGM3 (N=5)

1.54-5.38

10.9-18.2

0.06-0.25

GNE myopathy (N=12)

0.85-1.66

8.1-16.7

0.065-0.112

COG4

1.23

6.95

0.176

COG7 p1

0.80

7.67

0.105

COG7 p2

0.73

6.33

0.116

Mixed type I and II

1.30

19.02

0.068

28. Inject samples on the LC-MS/MS machine (calibrators first, followed by normal control, positive control, and then patient samples) following the settings below: LC operating parameters: Gradient method: Binary Mobile Phase A: 98% water-2% acetonitrile-0.2% formic acid (v/v/v) Mobile Phase B: 98% acetonitrile-2% water-0.2% formic acid (v/v/v) Flow rate: 0.25 ml/min LC column: C18 Thermogold 3μm 2.0 × 100 mm column Injection volume: 10 μl Run time: 25 min MS source parameters: Source: ES+ CUR: 25 CAD: Medium IS: 5500 TEM: 600 GS1: 55 GS2: 65 Multiple reaction monitoring (MRM) of analyte mass pairs, DP, CE, and CXP. Q1 (m/z)

Q3 (m/z)

Time (msec)

ID

DP (V)

CE (V)

CXP (V)

534.204 681.151 895.406

298.191 463.215 520.304

260 260 260

T antigen-PerMe-Na Raffinose-PerMe-Na ST-PerMe-Na

150 150 150

43 57 63

14 20 22

Analyze data 29. Using the LC-MS/MS analysis software, generate a new method and calibrate the data for the standard curve, normal control, positive control, and patient samples. 30. Calculate concentrations of T antigen and ST antigen, as well as the ratio of T/ST for each normal control, positive control, and patient samples as shown in Table 17.18.3. Glycosylation Analysis for Congenital Disorders of Glycosylation

17.18.18 Supplement 86

Current Protocols in Human Genetics

REAGENTS AND SOLUTIONS Use Milli-Q purified or higher-quality water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2D.

Acetic acid in methanol solution, 0.25 M Place 7.08 ml glacial acetic acid into 500-ml graduated cylinder. Add methanol to bring final volume to 500 ml. Mix well, and pour into a glass bottle. Store at room temperature for up to 1 year. Acetonitrile, 50% with 0.1% TFA solution Mix 5 ml acetonitrile and 5 ml Milli-Q water. Add 10 μl TFA to solution. Mix well, and store at room temperature for up to 1 year. AG 50W-X8 resin solution Add resin to a 500-ml bottle to about one-third volume. Add 300 to 400 ml water, agitate to mix, and wait for the resin to settle to the bottom. Pour off the supernatant carefully and repeat this step until the supernatant is clear. Keep the resin in 300 to 400 ml water. Store at room temperature for up to 1 year. Elution buffer (0.1 M glycine-acetic acid, pH 2.5) Dissolve 0.75 mg glycine in 80 ml water. Add 2 ml acetic acid. Mix well and adjust pH to 2.5 with concentrated HCl. Bring final volume to 100 ml with water. Store at 4°C for up to 1 year. Ethanolamine-hydrochloride, 1 M Take 1.208 ml ethanolamine, add 13.792 ml Milli-Q water (total 15 ml), and mix well. Adjust pH to 8.0 with 6 N HCl. Bring final volume to 20 ml. MOPS-NaOH, 0.1 M (pH 7.5) Weigh 4.185 g MOPS and dissolve in 180 ml Milli-Q water. Adjust pH to 7.5 with 1 N NaOH. Bring final volume to 200 ml with Milli-Q water. MOPS-NaOH, 10 mM (pH 7.5) Take 100 ml of 0.1 M MOPS-NaOH (pH 7.5), and add Milli-Q water to final volume of 1000 ml. N- and O-glycan MALDI matrix buffer (2,5-dihydroxybenzoic acid) Weigh 2.2 mg of 2,5-dihydroxybenzoic acid into 1.5-ml microcentrifuge tube, and add 200 μl of 50% methanol (11 mg/ml concentration). Add 4 μl of 0.05 M sodium acetate to make the final concentration 1 mM sodium acetate. Vortex and store at room temperature for up to 3 days. For the best crystal formation, use freshly made matrix buffer. Normal control Collect pooled plasma from healthy individuals (50 ml for each protocol). Ensure the mono-/ditransferrin ratio, N-glycan levels, and O-glycan levels fall within normal reference range. Aliquot and store at −80°C for up to 2 years. Positive control For transferrin: Spike 100 μg monoglycosylated transferrin into 100 μl normal plasma pool. Validate the mono-/ditransferrin ratio. For O-glycan: Spike 2 μl of 13 mM standard T antigen glycan into 1 ml normal plasma. (Target concentration is 25 μM.) Aliquot and store at −80°C. Positive control is stable for at least 2 years

Biochemical Genetics

17.18.19 Current Protocols in Human Genetics

Supplement 86

Sodium borohydride, 2 M in 0.1 M sodium hydroxide solution Dissolve 76 mg sodium borohydride powder in 1 ml of 0.1 M sodium hydroxide solution. Mix well, and use immediately. Transferrin MALDI matrix buffer (α-cyano-4-hydroxycinnamic acid) Weigh 2.0 mg α-cyano-4-hydroxycinnamic acid, and add 0.20 ml acetonitrile-TFA solution to make 10 mg/ml concentration. Vortex solution 1 min, and centrifuge 1 min at 3800 rpm, room temperature. Store at 4°C for up to 1 week. Washing solution (resin storage buffer, pH 7.5) 50 mM Tris-HCl, 0.15 M NaCl, and 0.02% sodium azide Weigh 0.1 g sodium azide and dissolve in 200 ml Milli-Q water. Add 25 ml of 1 M Tris-HCl, pH 7.5 stock solution. Add 18.75 ml of 4 M NaCl. Bring final volume to 500 ml with water. Store at room temperature for up to 1 year.

COMMENTARY Background Information

Glycosylation Analysis for Congenital Disorders of Glycosylation

Protein glycosylation is the process of adding various monosaccharides linked to either the amide group of asparagine (N-linked) or the hydroxyl group of serine or threonine (O-linked) (Freeze, 2006). The majority of CDG subtypes are caused by primary defects in N- and/or O-glycosylation pathways, which lead to defective glycoprotein biosynthesis. The biosynthesis of N-glycans, as well as Oglycans, can be divided into four stages: (1) the biosynthesis and activation of monosaccharides, (2) the entry of dolichol phosphate bound sugars into the endoplasmic reticulum and nucleotide sugars into the Golgi apparatus, (3) assembly of the nucleotide sugars or dolichol linked sugar onto N- or certain Olinked glycans in the endoplasmic reticulum, and (4) the processing of these glycans in the Golgi apparatus (Freeze, 2006). Defects in any of these four steps can lead to alterations of glycan structures on glycoproteins. It is estimated that >20% of all human proteins are glycosylated through N- or O-linkages (excluding O-GlcNAcylated proteins; Thiel et al., 2003). The broad symptomatology of glycosylation disorders and the tremendous structural diversity of glycoconjugates renders the identification of glycosylation defects challenging. The N-glycan profile of total plasma or serum total glycoprotein directly measures the structure and composition of N-linked glycans. This makes it probably the best tool to screen for type II CDGs biochemically. Although transferrin analysis by ESI-MS or electrophoresis, but not by MALDI-TOF, can pick up a portion of type II CDGs, the resolution and sensitivity of these analyses are low; and, results are often difficult to interpret.

It has not been well-recognized that many type I CDG subtypes not only have hypoglycosylation of glycoproteins, but also have abnormal glycan patterns that are related to the specific blockage in the pathway. One example is the identification of mannose deficient N-linked tetrasaccharide (Sail1 Gal1 GlcNAc2 ) in PMM2-CDG, MPI-CDG, and ALG1-CDG. As PMM2-CDG, MPI-CDG, and ALG1-CDG comprise nearly 70% of patients with type I CDG, these diagnostic biochemical markers in N-glycan profiles greatly facilitate the diagnoses of the majority of type I CDG patients. As both CDT and N-glycan profiles are currently available clinically, both should be considered as first-tier screening tests for CDGs. O-glycan profiling and quantification of T and ST antigens are diagnostic tools for combined N-linked and O-linked glycosylation disorders as well as GNE-CDG. The sensitivity of O-glycan analysis could be higher than other glycosylation measurements, particularly in the case of GNE-CDG (Leoyklang et al., 2014); however, it is also prone to secondary effects from other conditions, such as severe liver or kidney diseases. Therefore, O-linked glycan analysis could be used as a screening tool. Similar to O-glycan analysis, it is known that hypoglycosylation of transferrin also occurs in individuals with chronic alcohol abuse (Maenhout et al., 2014). It was also previously reported that a mild increase of N-glycans lacking terminal galactose could be seen in patients with chronic inflammatory diseases (Lauc et al., 2013), which may be related to increased secretion of immunoglobulins into the blood. We have also noted that increased fucosylation of N- or O-glycans are relatively common in sick patients and could

17.18.20 Supplement 86

Current Protocols in Human Genetics

be related to infections or inflammatory diseases. Hemolytic-uremic syndrome (HUS) is another condition that could lead to abnormal glycosylation in the blood. In HUS, isolated hyposialylation of N-linked glycans is usually seen due to the infection of neuraminidase secreting microorganisms in the blood (Guillard et al., 2011).

This will allow the efficiency of β-elimination to be observed. If the amount of transferrin spotted on the plate is too low, the ratio between different transferrin glycoforms will vary, and borderline changes could be missed in such cases. Therefore, it is important to monitor the concentration and purity of transferrin.

Critical Parameters

Anticipated Results

Permethylation is an important step for both N- and O-glycan preparation. We have found that the liquid-liquid permethylation method is ideal for protecting multiple sialylated species. As the abundance of multiantennary sialylated N-glycans in the blood is relatively low and their levels are important in detecting PGM3-CDG, we chose the liquid-liquid permethylation method for Nglycan preparation. On the other hand, we have found that solid phase permethylation is better than liquid-liquid permethylation in preserving asialylated small glycans, such as T antigens, in O-glycan profiles. Therefore, solid phase permethylation is a better method for O-glycan preparation overall. Because ambient moisture could interfere with permethylation reactions, a small amount of water (

Glycosylation Analysis for Congenital Disorders of Glycosylation.

Congenital disorders of glycosylation (CDG) are a group of diseases with highly variable phenotypes and inconsistent clinical features. Since the firs...
874KB Sizes 0 Downloads 8 Views