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Glycoform analysis of recombinant and human immunodeficiency virus envelope protein gp120 via higher energy collisional dissociation and spectral-aligning strategy.

Yang W, Shah P, Toghi Eshghi S, Yang S, Sun S, Ao M, Rubin A, Jackson JB, Zhang H - Anal. Chem. (2014)

Bottom Line: This challenge promoted us to employ a Q-Exactive mass spectrometer to identify low abundant glycopeptides from virion-associated gp120.We further revealed that except for Asn276 in the C2 region, glycans were processed to contain both high mannose and hybrid/complex glycans; an additional four N-linked glycosites were decorated with high mannose type.Core 1 O-linked glycan Gal1GalNAc1 was seen for the O-linked glycosite at Thr499.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine, Johns Hopkins University , 1550 Orleans Street , Baltimore, Maryland 21205, United States.

ABSTRACT
Envelope protein gp120 of human immunodeficiency virus (HIV) is armored with a dense glycan shield, which plays critical roles in envelope folding, immune-evasion, infectivity, and immunogenicity. Site-specific glycosylation profiling of recombinant gp120 is very challenging. Therefore, glycoproteomic analysis of native viral gp120 is still formidable to date. This challenge promoted us to employ a Q-Exactive mass spectrometer to identify low abundant glycopeptides from virion-associated gp120. To search the HCD-MS data for glycopeptides, a novel spectral-aligning strategy was developed. This strategy depends on the observation that glycopeptides and the corresponding deglycosylated peptides share very similar MS/MS pattern in terms of b- and y-ions that do not contain the site of glycosylation. Moreover, glycopeptides with an identical peptide backbone show nearly resembling spectra regardless of the attached glycan structures. For the recombinant gp120, this "copy-paste" spectral pattern of glycopeptides facilitated identification of 2224 spectra using only 18 spectral templates, and after precursor mass correction, 1268 (57%) spectra were assigned to 460 unique glycopeptides accommodating 19 N-linked and one O-linked glycosylation sites (glycosites). Strikingly, we were able to observe five N- and one O-linked glycosites in native gp120. We further revealed that except for Asn276 in the C2 region, glycans were processed to contain both high mannose and hybrid/complex glycans; an additional four N-linked glycosites were decorated with high mannose type. Core 1 O-linked glycan Gal1GalNAc1 was seen for the O-linked glycosite at Thr499. This direct observation of site-specific glycosylation of virion-derived gp120 has implications in HIV glycobiology and vaccine design.

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Related in: MedlinePlus

MS/MSspectra of O-linked glycopeptides demonstrating the applicabilityof spectral-aligning strategy for identification of O-linked glycopeptides.Peptide IEPLGVAPT499KAK in different glycoforms Gal1GalNAc1 in (A) and NeuAc1Gal1GlcNAc1GalNAc1 in (B) are aligned to show a similar pattern of peaks.
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fig3: MS/MSspectra of O-linked glycopeptides demonstrating the applicabilityof spectral-aligning strategy for identification of O-linked glycopeptides.Peptide IEPLGVAPT499KAK in different glycoforms Gal1GalNAc1 in (A) and NeuAc1Gal1GlcNAc1GalNAc1 in (B) are aligned to show a similar pattern of peaks.

Mentions: O-linked glycopeptidescontain carbohydrate(s) attaching to Seror Thr, whose HCD MS/MS spectra could differ from that of its N-linkedcounterparts. To evaluate the applicability of our strategy for identificationof O-linked glycopeptides, experimentally identified peptides IEPLGVAPT499K and IEPLGVAPT499KAK were used to generate theoreticalb- and y-ions. Again filtering the presence of peptide b- and y-ionscoupled with inspecting for peptide and Y-ions against 341 putativeglycopeptide spectra containing at least four y+-ions,we identified O-linked glycopeptides with glycans, Gal1GalNAc1, and other O-linked glycopeptides, one of whichwas with glycan NeuAc1Gal1GlcNAc1GalNAc1 (Figure 3).In the MS/MS spectra of O-linked glycopeptides, the peptide b- andy-ions were likely to appear because, possibly, the O-linked glycanswere easily cleaved off from the peptide moiety at the glycosidicbond resulting in intensive fragmentation of the peptide backbone(Figure 3). In addition, it was noticed thatpeak intensity of Y1 and other Y-ions was lower to b- and y-ions andcould even be absent under the NCE we applied (Figure 3). These observations suggested that oxonium ion HexNAc at m/z 204 and peptide b- and y-ions werehighly relevant for identification of O-linked glycopeptides, andthe spectral-aligning strategy was applicable to O-linked glycopeptides.Finally, a total number of 105 MS/MS spectra were identified fromO-linked glycopeptides bearing peptide backbones of either the IEPLGVAPT499KAK or the IEPLGVAPT499K. Using the samecriteria, only one putative O-linked glycopeptide was matched in theplatelet raw file scoring approximately 0.6% FDR, and this spectrawas dissimilar to its verified glycopeptides from recombinant gp120.


Glycoform analysis of recombinant and human immunodeficiency virus envelope protein gp120 via higher energy collisional dissociation and spectral-aligning strategy.

Yang W, Shah P, Toghi Eshghi S, Yang S, Sun S, Ao M, Rubin A, Jackson JB, Zhang H - Anal. Chem. (2014)

MS/MSspectra of O-linked glycopeptides demonstrating the applicabilityof spectral-aligning strategy for identification of O-linked glycopeptides.Peptide IEPLGVAPT499KAK in different glycoforms Gal1GalNAc1 in (A) and NeuAc1Gal1GlcNAc1GalNAc1 in (B) are aligned to show a similar pattern of peaks.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215848&req=5

fig3: MS/MSspectra of O-linked glycopeptides demonstrating the applicabilityof spectral-aligning strategy for identification of O-linked glycopeptides.Peptide IEPLGVAPT499KAK in different glycoforms Gal1GalNAc1 in (A) and NeuAc1Gal1GlcNAc1GalNAc1 in (B) are aligned to show a similar pattern of peaks.
Mentions: O-linked glycopeptidescontain carbohydrate(s) attaching to Seror Thr, whose HCD MS/MS spectra could differ from that of its N-linkedcounterparts. To evaluate the applicability of our strategy for identificationof O-linked glycopeptides, experimentally identified peptides IEPLGVAPT499K and IEPLGVAPT499KAK were used to generate theoreticalb- and y-ions. Again filtering the presence of peptide b- and y-ionscoupled with inspecting for peptide and Y-ions against 341 putativeglycopeptide spectra containing at least four y+-ions,we identified O-linked glycopeptides with glycans, Gal1GalNAc1, and other O-linked glycopeptides, one of whichwas with glycan NeuAc1Gal1GlcNAc1GalNAc1 (Figure 3).In the MS/MS spectra of O-linked glycopeptides, the peptide b- andy-ions were likely to appear because, possibly, the O-linked glycanswere easily cleaved off from the peptide moiety at the glycosidicbond resulting in intensive fragmentation of the peptide backbone(Figure 3). In addition, it was noticed thatpeak intensity of Y1 and other Y-ions was lower to b- and y-ions andcould even be absent under the NCE we applied (Figure 3). These observations suggested that oxonium ion HexNAc at m/z 204 and peptide b- and y-ions werehighly relevant for identification of O-linked glycopeptides, andthe spectral-aligning strategy was applicable to O-linked glycopeptides.Finally, a total number of 105 MS/MS spectra were identified fromO-linked glycopeptides bearing peptide backbones of either the IEPLGVAPT499KAK or the IEPLGVAPT499K. Using the samecriteria, only one putative O-linked glycopeptide was matched in theplatelet raw file scoring approximately 0.6% FDR, and this spectrawas dissimilar to its verified glycopeptides from recombinant gp120.

Bottom Line: This challenge promoted us to employ a Q-Exactive mass spectrometer to identify low abundant glycopeptides from virion-associated gp120.We further revealed that except for Asn276 in the C2 region, glycans were processed to contain both high mannose and hybrid/complex glycans; an additional four N-linked glycosites were decorated with high mannose type.Core 1 O-linked glycan Gal1GalNAc1 was seen for the O-linked glycosite at Thr499.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine, Johns Hopkins University , 1550 Orleans Street , Baltimore, Maryland 21205, United States.

ABSTRACT
Envelope protein gp120 of human immunodeficiency virus (HIV) is armored with a dense glycan shield, which plays critical roles in envelope folding, immune-evasion, infectivity, and immunogenicity. Site-specific glycosylation profiling of recombinant gp120 is very challenging. Therefore, glycoproteomic analysis of native viral gp120 is still formidable to date. This challenge promoted us to employ a Q-Exactive mass spectrometer to identify low abundant glycopeptides from virion-associated gp120. To search the HCD-MS data for glycopeptides, a novel spectral-aligning strategy was developed. This strategy depends on the observation that glycopeptides and the corresponding deglycosylated peptides share very similar MS/MS pattern in terms of b- and y-ions that do not contain the site of glycosylation. Moreover, glycopeptides with an identical peptide backbone show nearly resembling spectra regardless of the attached glycan structures. For the recombinant gp120, this "copy-paste" spectral pattern of glycopeptides facilitated identification of 2224 spectra using only 18 spectral templates, and after precursor mass correction, 1268 (57%) spectra were assigned to 460 unique glycopeptides accommodating 19 N-linked and one O-linked glycosylation sites (glycosites). Strikingly, we were able to observe five N- and one O-linked glycosites in native gp120. We further revealed that except for Asn276 in the C2 region, glycans were processed to contain both high mannose and hybrid/complex glycans; an additional four N-linked glycosites were decorated with high mannose type. Core 1 O-linked glycan Gal1GalNAc1 was seen for the O-linked glycosite at Thr499. This direct observation of site-specific glycosylation of virion-derived gp120 has implications in HIV glycobiology and vaccine design.

Show MeSH
Related in: MedlinePlus