Structural Investigations of Glycoconjugates at High Sensitivity

Mechref, Yehia; Novotný, Miloš V.

doi:10.1021/cr0103017

Cited by 322 publications

(360 citation statements)

References 492 publications

(838 reference statements)

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“…While profiling glycopeptide compositions by MS has been previously reported, most examples found in the literature utilized tryptic digests or other site-specific enzymes [5,8,12,13,24]. These digestions facilitate glycopeptide analysis, because the peptide sequences produced are known a priori.…”

Section: Methods Designmentioning

confidence: 99%

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Glycoprotein profiling by electrospray mass spectrometry

Jiang

Desaire

Butnev

et al. 2004

J. Am. Soc. Mass Spectrom.

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This work compares several different methods of site-specific analysis of glycoproteins using electrospray mass spectrometry. The glycoprotein, oLH␣ (ovine luteinizing hormone, ␣-subunit) was chosen as an appropriate example protein for these studies because of its biological relevance and extreme microheterogeneity. More than 20 unique glycoforms were detected for this glycoprotein at the Asn 56 site of oLH␣. The carbohydrates present at this site affect receptor binding affinity, so understanding the great variety in the composition of these carbohydrates is important in studying ligand binding interactions. MS data was acquired on a quadrupole ion trap, a triple quadrupole, and a quadrupole time of flight mass spectrometer, and carbohydrate composition at the Asn 56 site of oLH␣ was determined using these instruments. Additionally, neutral loss and precursor ion scanning modes were also used to identify the glycoforms present, and these techniques were compared to the standard MS data. Of the three instruments compared in the study, the qTOF mass spectrometer achieved the lowest sample consumption, but all three instruments were useful in profiling the glycopeptide composition. (J Am Soc Mass Spectrom 2004, 15, 750 -758)

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Section: Methods Designmentioning

confidence: 99%

“…An alternate approach to analyzing the carbohydrates on glycoproteins involves digestion of the glycoprotein with endoproteases and characterizing the resulting glycopeptides [5][6][7][8]. The resultant glycopeptides may be analyzed by mass spectrometry with minimal clean-up.…”

mentioning

confidence: 99%

Glycoprotein profiling by electrospray mass spectrometry

Jiang

Desaire

Butnev

et al. 2004

J. Am. Soc. Mass Spectrom.

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“…These methodologies usually require significant labor and expertise in sample preparation and analysis of results. Most methods involve removal of the glycans from the protein [6][7][8]. Glycoanalysis of complex glycoproteins is therefore often time consuming, and may demand combination of several methods and high expertise [7].…”

Section: Introductionmentioning

confidence: 99%

A lectin array-based methodology for the analysis of protein glycosylation

Rosenfeld¹,

Bangio²,

Gerwig³

et al. 2007

Journal of Biochemical and Biophysical Methods

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Glycosylation is the most versatile and one of the most abundant protein modifications. It has a structural role as well as diverse functional roles in many specific biological functions, including cancer development, viral and bacterial infections, and autoimmunity. The diverse roles of glycosylation in biological processes are rapidly growing areas of research, however, Glycobiology research is limited by the lack of a technology for rapid analysis of glycan composition of glycoproteins. Currently used methods for glycoanalysis are complex, typically requiring high levels of expertise and days to provide answers, and are not readily available to all researcher.We have developed a lectin array-based method, Qproteome™ GlycoArray kits, for rapid analysis of glycosylation profiles of glycoproteins. Glycoanalysis is performed on intact glycoproteins, requiring only 4-6 h for most analysis types. The method, demonstrated in this manuscript by several examples, is based on binding of an intact glycoprotein to the arrayed lectins, resulting in a characteristic fingerprint that is highly sensitive to changes in the protein's glycan composition. The large number of lectins, each with its specific recognition pattern, ensures high sensitivity to changes in the glycosylation pattern. A set of proprietary algorithms automatically interpret the fingerprint signals to provide a comprehensive glycan profile output.

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“…MALDI tandem time of flight (MALDI-TOF/ TOF) is widely used partly because it has a higher dynamic range and has a high tolerance to salts and other contaminants. Besides, the complexity of data obtained in ESI-FTICR-MS, due to the presence of multiply charged ions and formation of salt adducts, greatly complicate data interpretation of complex glycopeptide mixtures [21]. However, unlike offline HPLC/MALDI-MS, online HPLC-ESI-FTICR-MS is a powerful technique for analyzing glycopeptide mixtures rapidly, efficiently, and in great detail in a single experiment [22].…”

mentioning

confidence: 99%

Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

Irungu

Zhang

et al. 2008

J. Am. Soc. Mass Spectrom.

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Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140⌬CFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis. (J

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Structural Investigations of Glycoconjugates at High Sensitivity

Cited by 322 publications

References 492 publications

Glycoprotein profiling by electrospray mass spectrometry

Glycoprotein profiling by electrospray mass spectrometry

A lectin array-based methodology for the analysis of protein glycosylation

Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

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