Fig 1.
MS spectra of the alkaline de-N-glycosylation products of MAB-1-L001.
(A) Products obtained from the reaction in 5% hydroxylamine; (B) products obtained from the reaction in 20% hydroxylamine. Both reactions were executed in hydroxylamine and 200 mM LiOH (final concentration) at 80°C for 1 h. The inserts show enlarged spectra of G0F(oxime and free glycan). G0F: fucosyl agalacto bi-antennary N-linked glycan; G1F: fucosyl monogalacto bi-antennary N-linked glycan. Glycan structures are depicted by using the CFG graphical notation for glycans (http://www.functionalglycomics.org/static/consortium/CFGnomenclature.pdf).
Fig 2.
Comparison between alkaline de-N-glycosylation at 80°C for 1 h and PNGase F digestion.
(A) HPLC chromatograms of PNGase F digestion (i), alkaline de-N-glycosylation without hydroxylamine (ii), alkaline de-N-glycosylation with 5% hydroxylamine (iii), and alkaline de-N-glycosylation with 20% hydroxylamine (iv). (B) Comparison of glycan recovery. Bar graph indicates sum of all glycan peak areas in the HPLC chromatograms. All chromatograms were obtained by injection of equal volumes of glycan solution prepared from the same quantity of glycoproteins. The numbered peaks were determined by MS and MS/MS. Glycan structures are depicted using CFG graphical notation for glycans (http://www.functionalglycomics.org/static/consortium/CFGnomenclature.pdf).
Table 1.
Assignment of the peaks in the HPLC chromatogram of N-glycans of MAB-1-L001 by MALDI-TOF MS.
Fig 3.
Time course of alkaline de-N-glycosylation with 20% hydroxylamine at 80°C.
(A) HPLC chromatograms of 2-AB-labeled N-linked glycans released by 0.5 h-reaction, 1 h-reaction, 2 h-reaction, and 4 h-reaction. (B) Comparison of glycan recovery. Bar graph indicates sum of all glycan peak areas in the HPLC chromatograms. All chromatograms were obtained by injection of equal volumes of glycan solution prepared from the same quantity of glycoproteins.
Fig 4.
Comparison of time courses of alkaline de-N-glycosylation at different temperatures.
Sum of all glycan peak areas in the HPLC chromatograms are plotted in red (80°C), orange (50°C), and green (25°C). The chromatograms are presented in S9 Fig. All chromatograms were obtained by injection of equal volumes of glycan solution prepared from the same quantity of glycoproteins.
Fig 5.
Comparison of HPLC profiles of 2-AB-labeled N-linked glycans released from bovine apo-transferrin by various conditions.
HPLC chromatograms of PNGase F digestion (i), alkaline de-N-glycosylation with 20% hydroxylamine at 80°C for 1 h (ii), and alkaline de-N-glycosylation with 20% hydroxylamine at 50°C for 1 h (iii). All chromatograms were obtained by injection of equal volumes of glycan solution prepared from the same quantity of glycoproteins. The numbered peaks were determined by MS. Glycan structures are depicted by using the CFG graphical notation for glycans (http://www.functionalglycomics.org/static/consortium/CFGnomenclature.pdf).
Fig 6.
Deacylation of N-linked glycans in apo-transferrin during alkaline de-N-glycosylation at 80°C for 1 h.
MS spectra of indicated fractions (i–iv) in the HPLC chromatogram of Fig 5-ii. Glycan structures are depicted by using the CFG graphical notation for glycans (http://www.functionalglycomics.org/static/consortium/CFGnomenclature.pdf).
Table 2.
Assignment of the peaks in HPLC chromatogram of N-glycans of apo-transferrin by MALDI-TOF MS.
Fig 7.
Comparison of HPLC profiles of 2-AB-labeled N-linked glycans released from HRP by various conditions.
(A) HPLC chromatograms of glycopeptidase A digestion (i), alkaline de-N-glycosylation with 20% hydroxylamine at 50°C for 1 h (ii), and alkaline de-N-glycosylation with 20% hydroxylamine at 80°C for 1 h (iii). (B) Comparison of glycan recovery. Bar graph indicates sum of all glycan peak areas in the HPLC chromatograms. All chromatograms were obtained by injection of equal volumes of glycan solution prepared from the same quantity of glycoproteins. The numbered peaks were determined by MS and MS/MS and are summarized in Table 3. Glycan structures are depicted by using CFG graphical notation for glycans (http://www.functionalglycomics.org/static/consortium/CFGnomenclature.pdf).
Fig 8.
MS/MS spectrum of degraded peak 7 appearing during alkaline de-N-glycosylation of HRP at 80°C for 1 h.
Fig 9.
MSn spectrum of degraded peak 8 appearing during alkaline de-N-glycosylation of HRP at 80°C for 1 h.
Table 3.
Assignment of the peaks in HPLC chromatogram of N-glycans of HRP by MALDI-TOF MS.