Protein Protocols Protein Protocols

910 Hooker and James
at individual glycosylation sites by MALDI-MS (21–24). Sample (0.5 µL) and glycosidase
(0.5 µL), or a combination of glycosidases, are incubated at 30°C for 24 h and 0.5-µL
aliquots are removed for MALDI-MS analysis (see Note 3).
3.3. Glycosylation Analysis by ESI-MS
ESI-MS has been used to aid the analysis of glycosylation macro- and microheterogeneity
and proteolytic cleavage of the C-terminal in conjunction with information
obtained by HPLC and MALDI-MS of released oligosaccharides (25; see Note 4).
1. Spectra are obtained with a VG Quattro II triple quadrupole mass spectrometer having a
mass range for singly charged ions of 4000 Da (Fig. 3).
2. Lyophilized proteins are dissolved in 50% aqueous acetonitrile, 0.2% formic acid to a
concentration of 0.1 µg/µL and introduced into the electrospray source at 4 µL/min. The
mass-to-charge (m/z) range of 600–1800 Da are scanned at 10 s/scan and data are summed
for 3–10 min, depending on the intensity and complexity of the spectra. During each scan,
the sample orifice-to-skimmer potential (cone voltage) are scanned from 30 V at m/z 600
to 75 V at m/z 1800. The capillary voltage is set to 3.5 kV.
3. Mass scale calibration employ the multiply charged ion series from a separate introduction
of horse heart myoglobin (average molecular mass of 16,951.49). Molecular weights
are based on the following atomic weights of the elements: C = 12.011, H = 1.00794,
N = 14.00674, O = 15.9994, and S = 32.066 (see Note 5).
4. Background subtracted m/z data are processed by software employing a maximumentropy
(MaxEnt) based analysis to produce zero-charge protein molecular weight information
with optimum signal-to-noise ratio, resolution, and mass accuracy.
4. Notes
1. Attempts to separate IFN-γ with borate alone, as used by Landers et al. (26) for the
separation of ovalbumin glycoforms (26), were unsuccessful, because SDS is required to
disrupt the hydrogen-bonded dimers. Application of this technique to ribonuclease B and
fetuin met with variable success. The glycoprotein microheterogeneity of a monoclonal
antibody with a single glycosylation site has been mapped using a borate buffer at high
pH; the glycans were enzymatically or chemically cleaved and the resulting profile used
for testing batch-to-batch consistency in conjunction with MALDI-MS analysis (27).
2. MALDI-MS of free N-linked oligosaccharides, following chemical release with hydrazinolysis
or enzymatic release with an endoglycosidase such as PNGaseF, is also popular
as it requires no prior structural knowledge of the glycoprotein of interest and is ideal for
the analysis of underivatized populations of oligosaccharides. However, there is a loss of
glycosylation site-specific data. Enzymatic release of oligosaccharides is preferred where
an intact deglycosylated protein product is required, as N-glycan release by hydrazinolysis
may result in peptide bond cleavage and the oligosaccharide product requires reacetylation.
A drawback to the enzymatic release of oligosaccharides is that the presence of SDS is
often required to denature the glycoprotein and has to be removed prior to MALDI-MS
analysis. MALDI-MS has also been used for the analysis of IFN-γ glycoforms separated
by SDS-polyacrylamide gel electrophoresis (PAGE) (28).
3. Until recently, only desialylated oligosaccharides could be analyzed successfully
by MALDI-MS using 2,5-dihydroxybenzoic acid as matrix, as negatively charged sialic
acids interfere with the efficiency of ionization using this procedure (29,30). However,
advances in matrix mixtures and sample preparation schemes now promise to further
improve analytical protocols. For example, sialylated oligosaccharides have recently been