Direct Approach for Qualitative and Quantitative Characterization of Glycoproteins Using Tandem Mass Tags and an LTQ Orbitrap XL Electron Transfer Dissociation Hybrid Mass Spectrometer

Ye, Hongping; Boyne, Michael T.; Buhse, Lucinda F.; Hill, John C.

doi:10.1021/ac3026465

Cited by 47 publications

(73 citation statements)

References 43 publications

(60 reference statements)

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“…For glycopeptides with attached glycans, oxonium ion intensities were observed to be higher than the intensities of the iTRAQ reporter ions, and the diagnostic oxonium ions were the base peak in MS/MS. In previous studies, a similar phenomenon was observed when tandem mass tags (TMT) were used for the analysis of bovine fetuin/glycosidic bonds were preferentially cleaved versus amides in HCD fragmentation (23,24). To extract the MS/MS spectra of glycopeptides from the global proteomic data, we required the oxonium ion intensities to be greater than the highest iTRAQ reporter ion intensity.…”

Section: Resultsmentioning

confidence: 82%

Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation*

Shah

Wang

Yang

et al. 2015

Molecular & Cellular Proteomics

113

123

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Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two-dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in fucosylation could be caused by the detected changes in enzymes belonging to the glycan biosynthesis pathways of protein fucosylation observed in our proteomic analysis. The altered protein fucosylation forms have great potential in aiding our understanding of castration resistance and may lead to the development of novel therapeutic approaches and specific detection strategies for prostate cancer. Molecular & Cellular

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Section: Resultsmentioning

confidence: 82%

Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation*

Shah

Wang

Yang

et al. 2015

Molecular & Cellular Proteomics

113

123

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“…Based on the online chromatogram, the target fractions were infused for MS analysis followed by subsequent higher-energy collisional dissociation (HCD) and ETD of the selected precursors. Detailed MS settings and parameters were identical to those previously described (28). Briefly, in-source dissociation at 92 V generated characteristic oxonium ions at m/z 204 which were further fragmented by a dedicated MS 3 event for highly sensitive and selective detection of the eluting glycosylated peptides (29).…”

Section: Liquid Chromatography-mass Spectrometrymentioning

confidence: 99%

“…The reported labeling procedure was developed using commercially available fetuin without any excipients (28). To achieve an optimized labeling procedure for the drug products, different approaches were investigated.…”

Section: Tmt Labeling Efficiency Of the Drug Productsmentioning

confidence: 99%

“…Recently, our group developed a qualitative and quantitative glycoprotein characterization method using isobaric labeling and liquid chromatography-mass spectrometry (LC-MS) on an LTQ Orbitrap XL instrument with electron transfer dissociation (ETD) capabilities (28). Because a unique multiplexed isobaric tandem mass tag (TMT) is used for each sample, up to six samples can be compared side by side in a single experiment.…”

Section: Introductionmentioning

confidence: 99%

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Direct Site-Specific Glycoform Identification and Quantitative Comparison of Glycoprotein Therapeutics: Imiglucerase and Velaglucerase Alfa

Hill²,

Gucinski

et al. 2014

AAPS J

Self Cite

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Abstract. Gaucher disease, the most common lysosomal metabolic disorder, can be treated with enzyme replacement therapy (ERT). Recombinant human glucocerebrosidase imiglucerase (Cerezyme ® ), produced in Chinese hamster ovary cells, has been used for ERT of Gaucher disease for 20 years. Another recombinant glucocerebrosidase velaglucerase alfa (VPRIV), expressed in a human fibroblast cell line, was approved by the US Food and Drug Administration in 2010. The amino acid sequence difference at residue 495 of these two products is well documented. The overall N-linked qualitative glycan composition of these two products has also been reported previously. Herein, employing our recently developed approach utilizing isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL electron transfer dissociation (ETD) hybrid mass spectrometer, the site-specific glycoforms of these products were identified with ETD and collision-induced dissociation (CID) spectra. The quantitative comparison of site-specific glycans was achieved utilizing higher-energy collisional dissociation (HCD) spectra with a NanoMate used as both a fraction collector and a sample introduction device. From the trypsin-digested mixture of these two products, over 90 glycopeptides were identified by accurate mass matching. In addition to those previously reported, additional glycopeptides were detected with moderate abundance. The relative amount of each glycoform at a specific glycosylation site was determined based on reporter signal intensities of the TMT labeling reagents. This is the first report of site-specific simultaneous qualitative and quantitative comparison of glycoforms for Cerezyme ® and VPRIV. The results demonstrate that this method could be utilized for biosimilarity determination and counterfeit identification of glycoproteins.

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“…[34][35][36][37][38] This general behavior has proven very valuable, as application of two complementary methods can enable thorough characterization of glycopeptide composition and structure. [39][40][41][42][43] Unsurprisingly, a number of other glycopeptide ion dissociation pathways have been observed which do not fall within the domain of strictly complementarity vibrational activation/dissociation and electron transfer/ capture dissociation. [44][45][46][47] This includes numerous examples of vibrational activation/dissociation methods providing information not limited to the carbohydrate group, but extending to the amino acid sequence of the peptide group as well.…”

Section: Introductionmentioning

confidence: 99%

Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment

Kolli

Dodds

2014

Analyst

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"Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment" (2014). Faculty Publications --Chemistry Department. 68. http://digitalcommons.unl.edu/chemfacpub/68Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment † Venkata Kolli and Eric D. Dodds * Tandem mass spectrometry (MS/MS) of glycopeptides stands among the principal analytical approaches for assessing protein glycosylation in a site-specific manner. The aims of such experiments are often to determine the monosaccharide connectivity of the glycan, the amino acid sequence of the peptide, and the site of glycan attachment. This level of detail is often difficult to achieve using any single ion dissociation method; however, precedent does exist for use of collision-induced dissociation (CID) to establish either the connectivity of the oligosaccharide or the sequence of the polypeptide depending upon the applied collision energy. Unfortunately, the relative energy requirements for glycan and peptide cleavage have not been thoroughly characterized with respect to specific physicochemical characteristics of the precursor ions. This report describes case studies on the energy-resolved CID pathways of model tryptic glycopeptides derived from Erythrina cristagalli lectin and bovine ribonuclease B. While glycopeptide ions having disparate physical and chemical characteristics shared strikingly similar qualitative responses to increasing vibrational energy deposition, the absolute collision energies at which either glycan or peptide fragmentations were accessed varied substantially among the precursor ions examined. Nevertheless, these data suggest that the energy requirements for peptide and glycan cleavage may be somewhat predictable based on characteristics of the precursor ion. The practical usefulness of these observations was demonstrated through implementation of online collision energy modulation such that both glycan and peptide fragmentation were captured in the same spectrum, providing near-exhaustive glycopeptide characterization in a single experiment. Overall, these results highlight the potential to further extend the capabilities of CID in the context of glycoproteomics.

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Direct Approach for Qualitative and Quantitative Characterization of Glycoproteins Using Tandem Mass Tags and an LTQ Orbitrap XL Electron Transfer Dissociation Hybrid Mass Spectrometer

Cited by 47 publications

References 43 publications

Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation*

Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation*

Direct Site-Specific Glycoform Identification and Quantitative Comparison of Glycoprotein Therapeutics: Imiglucerase and Velaglucerase Alfa

Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment

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