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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

Representative of MS/MS spectra for identification of N-linkedglycopeptides using HCD spectral-aligning strategy. (A) MS/MS spectrumof deglycosylated peptide SVD276FTDNAK provided theinformation on experimentally identified b- and y-ions. (B) MS/MSspectrum of glycopeptide with matched pattern of b- and y-ions tothat of deglycosylated peptide was identified. Additional b-, y-,and Y-ions facilitate identification of the glycopeptide SVN276FTDNAK with Man7GlcNAc2, whichwas used as a spectral template to identify glycopeptides with thesame peptide backbone but different glycan Man5GlcNAc4 in (C).
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fig2: Representative of MS/MS spectra for identification of N-linkedglycopeptides using HCD spectral-aligning strategy. (A) MS/MS spectrumof deglycosylated peptide SVD276FTDNAK provided theinformation on experimentally identified b- and y-ions. (B) MS/MSspectrum of glycopeptide with matched pattern of b- and y-ions tothat of deglycosylated peptide was identified. Additional b-, y-,and Y-ions facilitate identification of the glycopeptide SVN276FTDNAK with Man7GlcNAc2, whichwas used as a spectral template to identify glycopeptides with thesame peptide backbone but different glycan Man5GlcNAc4 in (C).

Mentions: In the HCD MS/MS spectra of glycopeptides, becausemost glycan structures are fragmented to oxonium ions during MS/MS,the b-, y- and Y-ions appear to be nearly identical among glycopeptideswith the same peptide backbone regardless of attached glycan structures.As illustrated, first the peptide b- and y-ions of deglycosylatedpeptide SVD276FTDNAK were used to identify the glycopeptidehaving the peptide backbone SVN276FTDNAK and glycan Man7GlcNAc2 (Figure 2A,B). Additionalglycopeptide with the same peptide backbone but different glycan,Man5GlcNAc4, was then identified (Figure 2C). Specifically, in the comparison between theMS/MS of the two glycopeptides (Figure 2B,C),the three components, including (i) the oxonium ions at m/z 204, 366, (ii) y-ions at m/z 332 (y3+), 447 (y4+), 548 (y5+), 695 (y6+) from spectrum of deglycosylated peptide and extra b- andy-ions at m/z 849 (b8+), 809 (y7+) from spectrum of glycopeptide,and (iii) peptide ion at m/z 995and Y-ions from peptide with cross-ring cleavage of GlcNAc at m/z 1079 to peptide with Man4GlcNAc2 at m/z 2049, were matched (Figure 2B,C). One ofthe differences in these two MS/MS spectra was the detection of peptidewith Man5GlcNAc2 at m/z 2213 in the MS/MS spectrum of glycopeptide with Man7GlcNAc2, as well as the peptide with Man3GlcNAc3 at m/z 2091and Man4GlcNAc3 at m/z 2252 in the MS/MS spectrum of glycopeptide with Man5GlcNAc4 (Figure 2B,C). Thesedifferential Y-ions in different MS/MS spectra of glycopeptides could,in part, facilitate the plausible assignment of the glycan composition.Nevertheless, HCD fragmentation of glycopeptides have been shown toimprove the number and intensity of peptide b- and y-ions over thoseobserved in CID glycopeptide fragmentation spectra, which shows glycanfragmentation predominantly.30 Therefore,we were able to use b-, y-, and Y-ions to cross-match glycopeptidesas illustrated in Figure 2. Using b-, y-, peptide,and/or Y-ions in the verified MS/MS spectral template of glycopeptidesto filter against the list of 9544 putative glycopeptide spectra,we obtained 2620 MS/MS spectra for putative glycopeptides. Finally,we manually verified a total number of 2119 MS/MS spectra of N-linkedglycopeptides of gp120. We tried to estimate tentative false discoveryrate (FDR) using a raw file analyzing platelet glycopeptides thathad 18 057 oxonium-ion-containing spectra. Use the same b-and y-ions from gp120 deglycosylated peptides and filtering criteria,81 spectra were matched to score a tentative FDR of 2.6%. Manual inspectionshowed that those 81 MS/MS spectra were not similar to spectral templatesfrom recombinant gp120, suggesting that manual inspection was necessaryto remove the false-positives. Detail of the tentative FDR estimationand discussion is described in Supporting Information.


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)

Representative of MS/MS spectra for identification of N-linkedglycopeptides using HCD spectral-aligning strategy. (A) MS/MS spectrumof deglycosylated peptide SVD276FTDNAK provided theinformation on experimentally identified b- and y-ions. (B) MS/MSspectrum of glycopeptide with matched pattern of b- and y-ions tothat of deglycosylated peptide was identified. Additional b-, y-,and Y-ions facilitate identification of the glycopeptide SVN276FTDNAK with Man7GlcNAc2, whichwas used as a spectral template to identify glycopeptides with thesame peptide backbone but different glycan Man5GlcNAc4 in (C).
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Related In: Results  -  Collection

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fig2: Representative of MS/MS spectra for identification of N-linkedglycopeptides using HCD spectral-aligning strategy. (A) MS/MS spectrumof deglycosylated peptide SVD276FTDNAK provided theinformation on experimentally identified b- and y-ions. (B) MS/MSspectrum of glycopeptide with matched pattern of b- and y-ions tothat of deglycosylated peptide was identified. Additional b-, y-,and Y-ions facilitate identification of the glycopeptide SVN276FTDNAK with Man7GlcNAc2, whichwas used as a spectral template to identify glycopeptides with thesame peptide backbone but different glycan Man5GlcNAc4 in (C).
Mentions: In the HCD MS/MS spectra of glycopeptides, becausemost glycan structures are fragmented to oxonium ions during MS/MS,the b-, y- and Y-ions appear to be nearly identical among glycopeptideswith the same peptide backbone regardless of attached glycan structures.As illustrated, first the peptide b- and y-ions of deglycosylatedpeptide SVD276FTDNAK were used to identify the glycopeptidehaving the peptide backbone SVN276FTDNAK and glycan Man7GlcNAc2 (Figure 2A,B). Additionalglycopeptide with the same peptide backbone but different glycan,Man5GlcNAc4, was then identified (Figure 2C). Specifically, in the comparison between theMS/MS of the two glycopeptides (Figure 2B,C),the three components, including (i) the oxonium ions at m/z 204, 366, (ii) y-ions at m/z 332 (y3+), 447 (y4+), 548 (y5+), 695 (y6+) from spectrum of deglycosylated peptide and extra b- andy-ions at m/z 849 (b8+), 809 (y7+) from spectrum of glycopeptide,and (iii) peptide ion at m/z 995and Y-ions from peptide with cross-ring cleavage of GlcNAc at m/z 1079 to peptide with Man4GlcNAc2 at m/z 2049, were matched (Figure 2B,C). One ofthe differences in these two MS/MS spectra was the detection of peptidewith Man5GlcNAc2 at m/z 2213 in the MS/MS spectrum of glycopeptide with Man7GlcNAc2, as well as the peptide with Man3GlcNAc3 at m/z 2091and Man4GlcNAc3 at m/z 2252 in the MS/MS spectrum of glycopeptide with Man5GlcNAc4 (Figure 2B,C). Thesedifferential Y-ions in different MS/MS spectra of glycopeptides could,in part, facilitate the plausible assignment of the glycan composition.Nevertheless, HCD fragmentation of glycopeptides have been shown toimprove the number and intensity of peptide b- and y-ions over thoseobserved in CID glycopeptide fragmentation spectra, which shows glycanfragmentation predominantly.30 Therefore,we were able to use b-, y-, and Y-ions to cross-match glycopeptidesas illustrated in Figure 2. Using b-, y-, peptide,and/or Y-ions in the verified MS/MS spectral template of glycopeptidesto filter against the list of 9544 putative glycopeptide spectra,we obtained 2620 MS/MS spectra for putative glycopeptides. Finally,we manually verified a total number of 2119 MS/MS spectra of N-linkedglycopeptides of gp120. We tried to estimate tentative false discoveryrate (FDR) using a raw file analyzing platelet glycopeptides thathad 18 057 oxonium-ion-containing spectra. Use the same b-and y-ions from gp120 deglycosylated peptides and filtering criteria,81 spectra were matched to score a tentative FDR of 2.6%. Manual inspectionshowed that those 81 MS/MS spectra were not similar to spectral templatesfrom recombinant gp120, suggesting that manual inspection was necessaryto remove the false-positives. Detail of the tentative FDR estimationand discussion is described in Supporting Information.

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