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Characterization Quality Control

Glycan Analysis MALDImini-1

N-Linked Glycan Analysis Using MALDImini-1 benefits

Structural Analysis and Identification of Sialyl Linkage Isomers Cell Line Optimization
click here • Compact size and simple configuration allows installation in confined spaces.
• Samples can be measured immediately at the same location they are prepared.
Operating Principle and Features 5 μL of commercial serum were denatured and reduced by SDS and
DTT. N-linked glycans were cleaved from glycoproteins by adding • Suitable for a wide range of applications, from measuring the molecular weights of
Conventional MS mass spectrometers are large and require peripheral PNGaseF (Peptide-N-glycosidase F) and letting it react for 18 hours at trace samples to structural analysis of complex molecules.
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equipment, but the space-saving MALDImini-1 fits in a space smaller 37 °C. 4 μL of the cleaved N-linked glycans were mixed directly with
than a piece of A3 size paper. The built-in vacuum pump means the 20 μL of the SALSA reaction solution and left to react for one hour at Culture
system can be operated anywhere regular 100 V AC power is available. room temperature. Later, a stabilizer reagent with a lactonic structure
An optional kit is also available for supplying gas from small gas was added and mixed, and then the GL-Tip Amide (GL Sciences) was
cartridges. Additionally, the MALDI ion source and Digital Ion Trap (DIT) used to remove the excess reagent. Also, the reducing terminal of the
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technology enable high-sensitivity MS and MS measurements across a glycan was labeled with 2-aminobenzamide. Samples prepared by the
wide mass range, even for trace sample quantities. process above were dripped onto a 0.5 μL sample plate and 0.5 μL of
a matrix (α-cyano-4-hydroxycinnamic acid solution containing sodium
Measurement Method chloride) was layered on top and dried. Then the MALDImini-1 was
used for MS analysis.
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Proteins include many acidic glycans that contain sialic acids, which
are analyzed by an HPLC or a mass spectrometer. HPLC generally Results Purification
requires using a reference glycan preparation and can have difficulty
discriminating between complex glycans down to sialic acid linkages, A wide variety of bifurcated, trifurcated, and other mainly glycan
for example. Mass spectrometers can have problems with unstable sialic composites were detected from the N-linked glycans derived from
acid residues being prone to desorption during analysis and an inability serum glycoproteins (Fig. 2). A comparison of two types of MS 2
to discriminate between forms with different binding isomers. Therefore, spectra for trifurcated glycans shows the glycans were detected 28
the sialic acid residues on N-linked glycans derived from serum were Da apart, which infers that there are two different glycans (α2,3-
stabilized using the sialic acid linkage specific alkylamidation method and α2,6-linked forms) in the same location. Also, given that MS 2
(SALSA method in Fig. 1) developed by Shimadzu. The compact results show a neutral loss mass equivalent to modified sialic acids,
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MALDImini-1 MALDI-DIT mass spectrometer was used for detection which is the basis for differentiating between sialic acid linkage forms,
and analysis. The SALSA method generates a mass difference between presumably the peak at m/z 3117.1 indicates a mixture of α 2,3-/α
linkage forms using a two-stage reaction that amidates α2,6-linked 2,6- forms and m/z 3145.2 indicates only the α2,6- form. MS analysis Characterization
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sialic acids with isopropylamine (iPA) and amidates α2,3-linked was used to determine the location on the glycan that generated the
sialic acids with methylamine (MA). That means MS can be used to fragment ion. For example, the fragment ion at m/z 720.0 in the MS 2
discriminate between sialic acid linked isomers that otherwise would results for a biantennary glycan at m/z 2448.1 cannot be explained Specifications
have identical masses. by successive desorption of glycans from the non-reducing terminal.
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However, a comparison of MS results for fragment ions that include Instrument MALDImini-1
α2,6-sialylation
sialic acid (m/z 2107.0) and do not include sialic acid (m/z 1783.9) Mass range m/z 650 to 70,000
iPA (+ 41 Da) iPA (+ 41 Da) indicates that the m/z 720.0 fragment ion is not detected in the latter
derivative derivative
results. That means the m/z 720.0 fragment ion is derived from the MS/MS mass range m/z 350 to 5,000
(Dm = 59 Da)
(Dm = 0 Da) （Dm = 28 Da）
three glycans on the non-reducing terminal that includes the sialic acid. Mass resolution > 4000 FWHM, [Glu1]-Fibrinopeptide B m/z 1570.68, scan speed 1000 Da/s Quality Control
1st reaction 2nd reaction
-18 Da derivative Sensitivity (MS) 1 fmol ([Glu1]-Fibrinopeptide B m/z 1570.68)
MA
Lactone form
500 fmol (BSA m/z 66,431)
α2,3-sialylation
Fig. 1 Overview of Sialic Acid Linkage Specific Alkylamidation (SALSA) Method Sensitivity (MS/MS) 10 fmol ([Glu1]-Fibrinopeptide B m/z 1570.68)
Mass accuracy Internal standard: < 200 ppm External standard: < 200 ppm (m/z 1,000 to 5,000)
% Int. ; Galactose (Gal) ; Fucose (Fuc) 2448.1
; N-acethylglucosamine (GlcNAc) MS n 1 ≤ n ≤ 3
; Mannose (Man)
; iPA-derivatized Neu5Ac (α2,6-linked)
; MA-derivatized Neu5Ac (α2,3-linked) Laser Medium: Nd:YLF Wavelength: 349 nm
3117.1
Sample plate Disposable FlexiMass-DS and stainless steel FlexiMass-SR (26 × 76 mm)
3145.2 Pharmacokinetics
1768.0 2116.1 Gases Argon and helium (min. 99 % at 40 to 60 kPa)
1605.7
Gas cartridge Regulator, He gas tubing, Ar gas tubing, and gas cartridge holder
Fig. 2 Mass Spectrum of N-Linked Glycans Derived from Serum Glycoproteins Power supply AC 100 to 240 V, 50/60 Hz, 960 VA
Conclusion Application Examples (Shimadzu Application News No.) Dimensions W 309 mm × D 385 mm × H 320 mm
Weight 25 kg
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Stabilization of sialic acids by the SALSA method and MS analysis • Protein identification Operating environment Temperature: 18 to 26 °C Humidity: 40 to 70 % max. (with no condensation) Others
by the MALDImini-1 system can be used to analyze the structure of • Structural analysis of glycans and glycopeptides (B100)
glycans, including the sialic acid linkage types. • Checking the mass of various molecules Software Saving data: Database using SQLite
Export file formats: mzML and mzXML
*1 Patent No. 06135710
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