GPQuest: A Spectral Library Matching Algorithm for Site-Specific Assignment of Tandem Mass Spectra to Intact N‑glycopeptides

Glycoprotein changes occur in not only protein abundance but also the occupancy of each glycosylation site by different glycoforms during biological or pathological processes. Recent advances in mass spectrometry instrumentation and techniques have facilitated analysis of intact glycopeptides in complex biological samples by allowing the users to generate spectra of intact glycopeptides with glycans attached to each specific glycosylation site. However, assigning these spectra, leading to identification of the glycopeptides, is challenging. Here, we report an algorithm, named GPQuest, for site-specific identification of intact glycopeptides using higher-energy collisional dissociation (HCD) fragmentation of complex samples. In this algorithm, a spectral library of glycosite-containing peptides in the sample was built by analyzing the isolated glycosite-containing peptides using HCD LC-MS/MS. Spectra of intact glycopeptides were selected by using glycan oxonium ions as signature ions for glycopeptide spectra. These oxonium-ion-containing spectra were then compared with the spectral library generated from glycosite-containing peptides, resulting in assignment of each intact glycopeptide MS/MS spectrum to a specific glycosite-containing peptide. The glycan occupying each glycosite was determined by matching the mass difference between the precursor ion of intact glycopeptide and the glycosite-containing peptide to a glycan database. Using GPQuest, we analyzed LC-MS/MS spectra of protein extracts from prostate tumor LNCaP cells. Without enrichment of glycopeptides from global tryptic peptides and at a false discovery rate of 1%, 1008 glycan-containing MS/MS spectra were assigned to 769 unique intact N-linked glycopeptides, representing 344 N-linked glycosites with 57 different N-glycans. Spectral library matching using GPQuest assigns the HCD LC-MS/MS generated spectra of intact glycopeptides in an automated and high-throughput manner. Additionally, spectral library matching gives the user the possibility of identifying novel or modified glycans on specific glycosites that might be missing from the predetermined glycan databases

Comparison of Enrichment Methods for Intact N- and O‑Linked Glycopeptides Using Strong Anion Exchange and Hydrophilic Interaction Liquid Chromatography

Heterogeneity of protein glycosylation poses great challenges for analysis that is key to understand structure and function of glycoproteins. Resolving this conundrum requires efficient and specific enrichment of intact glycopeptides for identification and quantitation. To this end, hydrophilic interaction chromatography (HILIC) has been commonly used to enrich intact N- and O-linked glycopeptides. However, its effectiveness to enrich isobarically labeled glycopeptides remains unclear. Here, we studied three different enrichment methods for enrichment of N- and O-linked glycopeptides. It was found that removal of N-glycans prior to enrichment of O-linked glycopeptides by HILIC improved identification of O-linked glycopeptides by mass spectrometry. We also compared the enrichment of intact N- and O-linked glycopeptides using other chromatography methods and found that using cartridges containing materials for strong anion exchange (SAX) chromatography increased yield and identification of N- and O-linked glycopeptides. The enrichment of O-linked glycopeptides was further improved when a Retain AX cartridge (RAX) was used. In particular, isobaric tag labeled glycopeptides after C18 desalting could be readily enriched by SAX and RAX cartridges but not by HILIC to enable quantitative glycoproteomics. It is anticipated that the use of SAX and RAX cartridges will facilitate broad applications of identifications and quantitation of glycoproteins

Alterations of HIV-1 envelope phenotype and antibody-mediated neutralization by signal peptide mutations

<div><p>HIV-1 envelope glycoprotein (Env) mediates virus attachment and entry into the host cells. Like other membrane-bound and secreted proteins, HIV-1 Env contains at its N terminus a signal peptide (SP) that directs the nascent Env to the endoplasmic reticulum (ER) where Env synthesis and post-translational modifications take place. SP is cleaved during Env biosynthesis but potentially influences the phenotypic traits of the Env protein. The Env SP sequences of HIV-1 isolates display high sequence variability, and the significance of such variability is unclear. We postulate that changes in the Env SP influence Env transport through the ER-Golgi secretory pathway and Env folding and/or glycosylation that impact on Env incorporation into virions, receptor binding and antibody recognition. We first evaluated the consequences of mutating the charged residues in the Env SP in the context of infectious molecular clone HIV-1 REJO.c/2864. Results show that three different mutations affecting histidine at position 12 affected Env incorporation into virions that correlated with reduction of virus infectivity and DC-SIGN-mediated virus capture and transmission. Mutations at positions 8, 12, and 15 also rendered the virus more resistant to neutralization by monoclonal antibodies against the Env V1V2 region. These mutations affected the oligosaccharide composition of N-glycans as shown by changes in Env reactivity with specific lectins and by mass spectrometry. Increased neutralization resistance and N-glycan composition changes were also observed when analogous mutations were introduced to another HIV-1 strain, JRFL. To the best of our knowledge, this is the first study showing that certain residues in the HIV-1 Env SP can affect virus neutralization sensitivity by modulating oligosaccharide moieties on the Env N-glycans. The HIV-1 Env SP sequences thus may be under selective pressure to balance virus infectiousness with virus resistance to the host antibody responses. (289 words)</p></div

Effects of SP mutations on JRFL Env expression, virus infectivity and reactivity to different MAbs.

<p>(A) Schematic representation of JRFL WT and four different SP mutations evaluated in this study. (B) Measurement of Env incorporation by Western blot. JRFL WT and mutant viruses were produced in transfected 293T cells, lysed, and analyzed by SDS-PAGE (4–20%) and Western blot. An anti-gp120 MAb cocktail (V3: 391/95-D, 694/98-D, 2219, 2558; C2: 847-D, 1006-30D; C5: 450-D, 670-D) and a p24 Gag MAb (91–5) were used to detect the relative levels of Env and Gag associated with virions. The ratios of Env/Gag were calculated. (C) The levels of Env incorporation into JRFL mutant virions relative to that of WT were calculated based on their Env/Gag ratios (WT value was set to 100%). *, p< 0.01 (ANOVA). (D) Infectivity of JRFL WT vs. mutant viruses in CD4+ TZM.bl cells exposed to titrated viruses with equivalent p24 contents. (E) Correlation of virus infectivity in CD4+ TZM.bl cells with Env incorporation into the virions by Spearman’s rank test. Virus infectivity was based on RLU produced upon infection with a fix amount of virus input (0.9 ng p24/ml).</p

Glycan Analysis by Isobaric Aldehyde Reactive Tags and Mass Spectrometry

Glycans play significant roles in physiological and pathological processes. Therefore, quantitative analysis of glycans from normal and disease specimens can provide insight into disease onset and progression. Relative glycan quantification usually requires modification of the glycans with either chromogenic or fluorogenic tags for optical measurement or isotopic tags for mass spectrometric analysis. Because of rapid advances in mass spectrometry (MS) instruments in resolution, sensitivity, and speed, MS-based methods have become increasingly popular for glycan analysis in the past decade. However, current isotopic tags for glycan labeling are mostly mass-shift tags generating mass differences in precursor ions for quantification, which can complicate mass spectra. In this study, we report the synthesis and characterization of isobaric aldehyde reactive tags (iARTs) for glycan quantification using tandem MS. We applied iARTs to the relative identification and quantification of glycans of gp120, a glycoprotein from human immunodeficiency virus. The results show that iARTs provide strong signals for glycan identification. Although we only show the synthesis and characterization of two iARTs reagents, iARTs can be readily expanded to six-plex tags for quantitative analysis of six samples concurrently

Effects of SP mutations on JRFL Env expression, virus infectivity and reactivity to different MAbs.

<p>(A) Schematic representation of JRFL WT and four different SP mutations evaluated in this study. (B) Measurement of Env incorporation by Western blot. JRFL WT and mutant viruses were produced in transfected 293T cells, lysed, and analyzed by SDS-PAGE (4–20%) and Western blot. An anti-gp120 MAb cocktail (V3: 391/95-D, 694/98-D, 2219, 2558; C2: 847-D, 1006-30D; C5: 450-D, 670-D) and a p24 Gag MAb (91–5) were used to detect the relative levels of Env and Gag associated with virions. The ratios of Env/Gag were calculated. (C) The levels of Env incorporation into JRFL mutant virions relative to that of WT were calculated based on their Env/Gag ratios (WT value was set to 100%). *, p< 0.01 (ANOVA). (D) Infectivity of JRFL WT vs. mutant viruses in CD4+ TZM.bl cells exposed to titrated viruses with equivalent p24 contents. (E) Correlation of virus infectivity in CD4+ TZM.bl cells with Env incorporation into the virions by Spearman’s rank test. Virus infectivity was based on RLU produced upon infection with a fix amount of virus input (0.9 ng p24/ml).</p

Analysis of REJO Env sugar moieties by lectin-probed Western blotting.

<p>The same amounts of Env from sucrose-pelleted virions were separated by SDS-PAGE (4–20%) under reducing conditions, blotted, and probed with an anti-gp120 MAb cocktail, an anti-gp41 MAb cocktail, and lectins (GNA, GRFT, and AAL). A) REJO WT virus produced in GnTI^- cells or in 293T cells in the presence vs absence of kifunensine (25μM) known to alter Env glycan compositions. B) Quantification of total density of Env bands from REJO WT shown in A). C) REJO WT and mutant viruses produced in 293T cells. D) Density measurements of the upper and lower Env bands as recognized by anti-gp120 MAbs and different lectins. Density analysis was done by Image Lab software. The two Env species with distinct molecular masses are indicated by red and green arrows.</p

Proteomic and glycoproteomic analyses of virion proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

<p>Sucrose-pelleted virions were denatured with 8M in1 M ammonia bicarbonate buffer. The denatured proteins were than prepared for trypsin digestion at 37°C overnight. The samples containing peptides were acidified pH = 3 and desalted using C18 SPE column. The C18 elute was dried in the speed-vac and then resuspended in 0.2% formic acid. The samples (1 μg) were then subjected to LC-MS/MS. Relative abundance of different glycoforms found on 2 identified glycopeptides from SP mutants vs WT were calculated and shown as ratio of mutant to WT.</p

Effects of SP mutations on REJO virus capture and transmission by DC-SIGN.

<p>(A) Parental Raji or Raji–DC-SIGN⁺ cells were incubated for 2 hours with WT or mutant viruses produced in 293T cells. Cells were washed extensively, and the amounts of p24 protein associated with the cells were measured by ELISA. (B) Parental Raji or Raji–DC-SIGN⁺ cells were incubated with WT and mutant viruses for 2 hours, washed to remove unbound viruses, and added to CD4+ TZM.bl cells. Viral transmission to the TZM-bl cells was determined by luciferase activity and calculated based on infection in TZM.bl cells without Raji cells as control (set to 100%). Background luciferase activity was determined in co-cultures without any virus. *, p< 0.05 as compared to WT (ANOVA). (C) Correlation of virus capture (top) and transmission (bottom) via DC-SIGN with the Env incorporation into the WT and mutant virions by Spearman’s rank test.</p

Neutralization of JRFL WT and mutant viruses by different MAbs and CD4-IgG2.

<p>Neutralization assays were performed for each of MAb-virus pairs as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006812#ppat.1006812.g002" target="_blank">Fig 2</a>. AUC values were calculated from titration curves. A) Titration curves of representative virus-MAb pairs. B) JRFL WT and mutant neutralization by MAbs targeting V2i, V3, V2q, and the CD4bs and by CD-IgG2. AUC values that decreased by >30% and had p<0.05 relative to WT are shown in red. Means and standard errors from two to three experiments are shown.</p