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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

Schematic representationof the HCD spectral-aligning strategyfor analysis of glycopeptides. Step 1, experimental b- and y-ionsof deglycosylated peptide backbones were recorded from sample treatedwith PNGaseF. Step 2, these b- and y-ions of the deglycosylated peptideswere used to screen the MS/MS spectra containing oxonium ions fromthe raw file for sample without PNGaseF treatment to identify putativeglycopeptides. The putative glycopeptides were further evaluated byprecursor ions, mass difference between glycopeptides and deglycosylatedpeptides, and Y-ions in the MS/MS spectra of the glycopeptides. Step3, the identified MS/MS spectra of glycopeptides were used as spectraltemplates to identify other glycopeptides with the same peptide backbonebut in different glycoforms.
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fig1: Schematic representationof the HCD spectral-aligning strategyfor analysis of glycopeptides. Step 1, experimental b- and y-ionsof deglycosylated peptide backbones were recorded from sample treatedwith PNGaseF. Step 2, these b- and y-ions of the deglycosylated peptideswere used to screen the MS/MS spectra containing oxonium ions fromthe raw file for sample without PNGaseF treatment to identify putativeglycopeptides. The putative glycopeptides were further evaluated byprecursor ions, mass difference between glycopeptides and deglycosylatedpeptides, and Y-ions in the MS/MS spectra of the glycopeptides. Step3, the identified MS/MS spectra of glycopeptides were used as spectraltemplates to identify other glycopeptides with the same peptide backbonebut in different glycoforms.

Mentions: We exploitedthe use of peptide b- and y-ions from deglycosylated peptides andmatched them to HCD MS/MS spectra containing oxonium ions to identifyglycopeptides. Next, the verified MS/MS spectra of glycopeptides wereused to cross-match all the other glycopeptides with the identicalpeptide backbones but varying in glycoforms using b-, y-, and/or Y-ions.The strategy was developed on the basis of the observation that thepresence of peptide b- and y-ions that do not contain a site of glycosylationwere relatively consistent between deglycosylated peptides and theirglycopeptides, and the MS/MS spectra of glycopeptides with the samepeptide backbones but different glycan moieties were nearly identical.Moreover, Y-ions could be absent in the HCD MS/MS spectra of O-linkedglycopeptides, suggesting that the use of peptide b- and y-ions wasa critical consideration to identify glycopeptides in the HCD-MS.Our search strategy included the following steps: (1) deglycosylatedpeptides and glycosylation sites were identified from PNGaseF treatedsample; (2) b- and y-ions of peptide backbone were determined experimentallyusing the deglycosylated peptide spectra; (3) MS/MS spectra containingoxonium ions were extracted from the raw file of the sample withoutPNGaseF treatment; (4) the b- and y-ions of the deglycosylated peptideswere used to match and filter the MS/MS spectra of glycopeptides;(5) peptide and Y-ions in the MS/MS spectra of glycopeptides wereused to further evaluate the identification; (6) the best-matchedMS/MS spectra of glycopeptides were used to cross-match the otherMS/MS spectra of glycopeptides with the same peptide backbones; (7)finally, the precursor m/z and theΔmass between the peptide moieties and glycopeptides were usedto determine the glycoforms (Figure 1).


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)

Schematic representationof the HCD spectral-aligning strategyfor analysis of glycopeptides. Step 1, experimental b- and y-ionsof deglycosylated peptide backbones were recorded from sample treatedwith PNGaseF. Step 2, these b- and y-ions of the deglycosylated peptideswere used to screen the MS/MS spectra containing oxonium ions fromthe raw file for sample without PNGaseF treatment to identify putativeglycopeptides. The putative glycopeptides were further evaluated byprecursor ions, mass difference between glycopeptides and deglycosylatedpeptides, and Y-ions in the MS/MS spectra of the glycopeptides. Step3, the identified MS/MS spectra of glycopeptides were used as spectraltemplates to identify other glycopeptides with the same peptide backbonebut in different glycoforms.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Schematic representationof the HCD spectral-aligning strategyfor analysis of glycopeptides. Step 1, experimental b- and y-ionsof deglycosylated peptide backbones were recorded from sample treatedwith PNGaseF. Step 2, these b- and y-ions of the deglycosylated peptideswere used to screen the MS/MS spectra containing oxonium ions fromthe raw file for sample without PNGaseF treatment to identify putativeglycopeptides. The putative glycopeptides were further evaluated byprecursor ions, mass difference between glycopeptides and deglycosylatedpeptides, and Y-ions in the MS/MS spectra of the glycopeptides. Step3, the identified MS/MS spectra of glycopeptides were used as spectraltemplates to identify other glycopeptides with the same peptide backbonebut in different glycoforms.
Mentions: We exploitedthe use of peptide b- and y-ions from deglycosylated peptides andmatched them to HCD MS/MS spectra containing oxonium ions to identifyglycopeptides. Next, the verified MS/MS spectra of glycopeptides wereused to cross-match all the other glycopeptides with the identicalpeptide backbones but varying in glycoforms using b-, y-, and/or Y-ions.The strategy was developed on the basis of the observation that thepresence of peptide b- and y-ions that do not contain a site of glycosylationwere relatively consistent between deglycosylated peptides and theirglycopeptides, and the MS/MS spectra of glycopeptides with the samepeptide backbones but different glycan moieties were nearly identical.Moreover, Y-ions could be absent in the HCD MS/MS spectra of O-linkedglycopeptides, suggesting that the use of peptide b- and y-ions wasa critical consideration to identify glycopeptides in the HCD-MS.Our search strategy included the following steps: (1) deglycosylatedpeptides and glycosylation sites were identified from PNGaseF treatedsample; (2) b- and y-ions of peptide backbone were determined experimentallyusing the deglycosylated peptide spectra; (3) MS/MS spectra containingoxonium ions were extracted from the raw file of the sample withoutPNGaseF treatment; (4) the b- and y-ions of the deglycosylated peptideswere used to match and filter the MS/MS spectra of glycopeptides;(5) peptide and Y-ions in the MS/MS spectra of glycopeptides wereused to further evaluate the identification; (6) the best-matchedMS/MS spectra of glycopeptides were used to cross-match the otherMS/MS spectra of glycopeptides with the same peptide backbones; (7)finally, the precursor m/z and theΔmass between the peptide moieties and glycopeptides were usedto determine the glycoforms (Figure 1).

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