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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
 n
 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,
 *1
 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
 3
 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.
 3
 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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