This document presents a case study analyzing O-glycans in a patient using LC-MS to determine if the patient has Congenital Disorders of Glycosylation (CDG). CDG are genetic disorders that affect the addition of sugar molecules to proteins. The study aims to analyze N- and O-glycans in clinically suspected subjects compared to controls. The LC-MS procedure, results, and conclusion that the technique can help detect CDG disorders are summarized.

1. ANALYSIS OF O-GLYCAN BY LC-MS
case study of CDG
Presented by- Kamble Mangal Y.
M pharm First year(pharmaceutical chemistry)
Roll no-545
DYPCOP, Akurdi, Pune.
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2. CONTENT
• Introduction
• Case study
• Symptoms
• Aim of this study
• Analytical technique
• Procedure
• Conclusion
• Reference
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3. What is CDG
• Congenital disorders of glycosylation (CDG) are a large group of rare
genetic disorders that affect the addition of sugar building blocks, called
glycans, to proteins in cells throughout the body.
• The addition of glycans to proteins is critical to the healthy function of
cells.
• People with CDG have a wide range of health problems because of this
chemical malfunction.
While glycosylation involves sugar, as glycans are compounds of sugar
molecules, CDG are not related to diabetes.
• Instead, CDG cause problems in the way sugar building blocks are
attached to proteins within and on the surfaces of cells, affecting how cells
in every part of the body function.
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4. • CDG were first reported in medical literature in 1980 by Dr. jaak jaeken &
colleagues.
• More than 80 different form of CDG have been identified.
• Several names have been used to describe these disorder including
carbohydrate deficient glycoprotein syndrome.
• CDG comprising is disorder of protein glycosylation are broken down into
two groups known as disorder of N-glycosylation & O-glycosylation.
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5. Symptoms of CDGs
•low muscle tone or floppiness (hypotonia)
•poor growth, failure to thrive
•developmental delays
•liver disease (hepatopathy) with elevated liver enzymes
•abnormal bleeding or blood clotting
• crossed eyes (strabismus)
•seizures
•stroke-like episodes
•heart problems, including fluid accumulation around the
heart.
•balance and coordination problems (ataxia)
•slurred speech (dysarthria)
•no puberty in girls
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6. The aims of this study are as follows:
• Analysis of N- and O-glycans in clinically suspected
subjects in comparison to normal healthy controls.
• Determination of the reference range for T-antigen and
ST-antigen.
• Emphasis of the need for early detection of CDGs in
suspected patients to avoid irreversible neurological
complications.
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7. Principle of Liquid Chromatography - Mass Spectrometry (LC-
MS)
• The LC-MS technology involves use of an HPLC, wherein the
individual components in a mixture are first separated followed by
ionization and separation of the ions on the basis of their
mass/charge ratio.
• The separated ions are then directed to a photo or electron multiplier
tube detector, which identifies and quantifies each ion.
• The ion source is an important component in any MS analysis, as
this basically aids in efficient generation of ions for analysis.
• To ionize intact molecules, the ion source could be APCI
(Atmospheric Pressure Chemical Ionization), ESI (Electronspray
Ionization).
• The choice of ion source also depends on the chemical nature of the
analyte of interest i.e. polar or non-polar.
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8. The major advantages of this technology include sensitivity,
specificity and precision as analysis is done at molecular level.
Instrumentation:
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9. Quantitative analysis of O-glycans by LC-MS/MS
Samples and materials used
Serum from 50 subjects and 50 controls were used.
The internal standard raffinose (1250 pmol/5 μL) and
disodium tetraborate were purchased from Lobachemie
(Mumbai, India). Ion exchange AG 50 W-X8 resin, Tantigen
standard, ST-antigen standard, anhydrous dimethyl
sulfoxide (DMSO), and iodomethane (CH3I)
were purchased from Sigma-Aldrich (St Louis, USA).
C18 stage tips .
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10. Procedure-
25 μL of internal standard and 65 μL of water were added to 10 μL of serum.
100 μL of freshly prepared sodium borate in NaOH was added, and the
mixture was incubated for 16 h in a water bath at 45 °C.
Then, 1.6 mL acetic acid in methanol was added dropwise to neutralize the reaction.
the solution was desalted using ion-exchange AG 50 W-X8 resin.
The separated glycans were vacuum evaporated until completely dry.
The glycan chains were then permethylated according to the method.
four NaOH pellets were crushed in 10 mL of anhydrous DMSO and 0.5 μL of water
to make a slurry.
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11. Then, 0.5 mL of the slurry was added along with 0.2 mL of CH3I to the
lyophilized glycan and vigorously shaken for 1 h.
The mixture was then extracted 5 times using a mixture of 200 μL of water and
600 μL of chloroform, and then, the chloroform phases were pooled and dried
under nitrogen for 30 min.
To quantify the free glycans in each sample, 20 μL of serum was diluted to
500 μL and centrifuged at 10,000 rpm for 10 min at 4 °C.
The concentration of the free glycans was then subtracted from that of
the sample.
Dried permethylated patient and control samples were reconstituted with
50 μL of methanol, purified through C18 stage tips.
finally analysed on a triple quad mass spectrometer using a 3-μm C18 column
(2 mm × 100 mm) and a 10 μL sample volume in positive ion mode. 11

12. The mobile phase consisted of 2 Buffers :
buffer A (1:0.1:99) acetonitrile: formic acid:water
buffer B (99:0.1:1) acetonitrile: formic acid: water
• flow rate of 0.25 ml/min.
• Gradient elution was used as follows:
1. from 0–20 min, 50 to 80% buffer B
2. from 20–28 min, 98% buffer B
3. from 28–39 min, 50% buffer B.
Calibration curves were constructed using 6 concentrations of T-
antigen and ST-antigen each.
The results are expressed as the concentration of T-antigen
and ST-antigen as well as the T/ST ratio.
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13. Analysis of T-antigen and ST-antigen by LC-MS/MS with a calibration curve of T-antigen
standard, b calibration curve of ST-antigen standard, c mean ± SD of T-antigen and d mean ±
SD of ST-antigen 13

14. Chromatograms obtained from LC-MS/MS analysis. a Normal healthy control. b A case of COG5-
CDG. Types of CDG are determined after molecular analysis
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15. • LC-MS/MS method is sensitive at m/z values less than 2000 and therefore was
suitable to study the small T-antigen with an MRM transition of m/z 534/298 and
ST-antigen with an MRM transition of m/z 895/520.
• LC-MS/MS is considered a “soft” ionization technique where the analysed
compound is not subjected to in-source fragmentation that can disrupt its structure.
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16. Conclusion:
In this study, we concluded that the combined analysis O-glycans using LC-MS/MS
can provide a solid foundation towards the detection of hypoglycosylation
in subjects suspected to have CDG.
Test should be performed to avoid missing the diagnosis of O-glycan disorders such
as the CDGs resulting from COG deficiency.
All detected CDG cases were confirmed by molecular analysis results of mutations
causing 4 different types of congenital disorders of glycosylation.
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17. Reference:
1)http://www.mirarehab.com/blog/case-study-on-a-girl-with-congenital-
disorder-of-glycosylation/
2) Amr Sobhi Gouda1, Azza Fahmy Elbaz1*, Thierry Dupré2, Ola Sayed
Mohamed Ali3, Maha Saad Zaki4 and Ekram Maher Fateen1, “N- and O-
glycan analysis for the detection of glycosylation disorders”.
3)https://www.thyrocare.com/LiquidChromatography#:~:text=Principle%20of
%20Liquid%20Chromatography%20-%20Mass,of%20their%20mass%2
4) https://rarediseases.org/rare-diseases/congenital-disorders-of-glycosylation/
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