An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF.
Different glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles stil...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2010-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC2994013?pdf=render |
| _version_ | 1828336295418527744 |
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| author | Piliang Hao Yan Ren Yongming Xie |
| author_facet | Piliang Hao Yan Ren Yongming Xie |
| author_sort | Piliang Hao |
| collection | DOAJ |
| description | Different glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles still exist in analyzing sialylated glycoproteins due to the lability and higher detection limit of acid glycans in MALDI-TOF/TOF analysis. Thus, we present an improved protocol here. Tryptic glycopeptides were separated and subjected to MALDI-TOF/TOF analysis, resulting in the identification of site-specific glycosylation of high-intensity glycopeptides. Sequential deglycosylation and desialylation were used to improve the identification of glycosylation sites and desialylated glycans. The site-specific glycosylation of large glycopeptides and low-intensity glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. By applying it to 2-DE separated human serum, the difference of N-glycosylation was successfully determined for α1-antitrypsin between different gel spots. |
| first_indexed | 2024-04-13T21:58:57Z |
| format | Article |
| id | doaj.art-1ef2cf27ab144c4dafea79299b0762ba |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T21:58:57Z |
| publishDate | 2010-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-1ef2cf27ab144c4dafea79299b0762ba2022-12-22T02:28:10ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-01-01511e1509610.1371/journal.pone.0015096An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF.Piliang HaoYan RenYongming XieDifferent glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles still exist in analyzing sialylated glycoproteins due to the lability and higher detection limit of acid glycans in MALDI-TOF/TOF analysis. Thus, we present an improved protocol here. Tryptic glycopeptides were separated and subjected to MALDI-TOF/TOF analysis, resulting in the identification of site-specific glycosylation of high-intensity glycopeptides. Sequential deglycosylation and desialylation were used to improve the identification of glycosylation sites and desialylated glycans. The site-specific glycosylation of large glycopeptides and low-intensity glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. By applying it to 2-DE separated human serum, the difference of N-glycosylation was successfully determined for α1-antitrypsin between different gel spots.http://europepmc.org/articles/PMC2994013?pdf=render |
| spellingShingle | Piliang Hao Yan Ren Yongming Xie An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF. PLoS ONE |
| title | An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF. |
| title_full | An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF. |
| title_fullStr | An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF. |
| title_full_unstemmed | An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF. |
| title_short | An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF. |
| title_sort | improved protocol for n glycosylation analysis of gel separated sialylated glycoproteins by maldi tof tof |
| url | http://europepmc.org/articles/PMC2994013?pdf=render |
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