Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity

Yang, Yang; Liu, Fan; Franc, Vojtěch; Halim, Liem Andhyk; Schellekens, Huub; Heck, Albert J. R.

doi:10.1038/ncomms13397

Cited by 145 publications

(233 citation statements)

References 55 publications

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“…This increased resolving power has enabled small differences in proteoforms, such as glycosylation on antibodies [53, 54] and phosphorylation on proteins and protein complexes [55, 56], to be more readily resolved. One striking example is the analysis of erythropoietin, whereby 236 glycan proteoforms were separated by m / z and identified [57]. As such, the Orbitrap mass analyzer is rapidly becoming on par with the Q-ToF for native protein MS analysis, with both instruments having their own strong niches today.…”

Section: Key Milestones In Native Msmentioning

confidence: 99%

Native Mass Spectrometry: What is in the Name?

Leney

Heck

2016

J. Am. Soc. Mass Spectrom.

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503

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Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein–protein and protein–ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as “native MS,” has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure–function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by “native MS,” when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. Graphical Abstractᅟ

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Section: Key Milestones In Native Msmentioning

confidence: 99%

Native Mass Spectrometry: What is in the Name?

Leney

Heck

2016

J. Am. Soc. Mass Spectrom.

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503

480

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“…In this mass calculation, we used the mass of the C9 backbone sequence lacking the N -terminal signal peptide, corrected by the mass shift induced by the 12 disulfide bonds present in C9 (−24 × 1.0079 Da). Compared to, for instance, chicken ovalbumin 31 and CHO derived erythropotein, 10 which we previously analyzed by high-resolution native mass spectrometry, the native mass spectra of C9 are remarkably less heterogeneous, especially since, according to previously published data, C9 has been shown to be C -mannosylated 22 at the TPS domain and N -glycosylated 19−21 at two sites in the MACPF domain.…”

Section: Resultsmentioning

confidence: 91%

Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation

2017

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The human complement C9 protein (∼65 kDa) is a member of the complement pathway. It plays an essential role in the membrane attack complex (MAC), which forms a lethal pore on the cellular surface of pathogenic bacteria. Here, we charted in detail the structural microheterogeneity of C9 purified from human blood serum, using an integrative workflow combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The proteoform profile of C9 was acquired by high-resolution native mass spectrometry, which revealed the co-occurrence of ∼50 distinct mass spectrometry (MS) signals. Subsequent peptide-centric analysis, through proteolytic digestion of C9 and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) measurements of the resulting peptide mixtures, provided site-specific quantitative profiles of three different types of C9 glycosylation and validation of the native MS data. Our study provides a detailed specification, validation, and quantification of 15 co-occurring C9 proteoforms and the first direct experimental evidence of O-linked glycans in the N-terminal region. Additionally, next to the two known glycosylation sites, a third novel, albeit low abundant, N-glycosylation site on C9 is identified, which surprisingly does not possess the canonical N-glycosylation sequence N-X-S/T. Our data also reveal a binding of up to two Ca2+ ions to C9. Mapping all detected and validated sites of modifications on a structural model of C9, as present in the MAC, hints at their putative roles in pore formation or receptor interactions. The applied methods herein represent a powerful tool for the unbiased in-depth analysis of plasma proteins and may advance biomarker discovery, as aberrant glycosylation profiles may be indicative of the pathophysiological state of the patients.

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“…We envisage that developments in ion mobility resolution will enable the isomeric discrimination and assignment of larger and more complicated glycopeptides. Additionally, high-resolution mass spectrometry of intact glycoproteins is highly informative in both occupancy and glycan composition which has been demonstrated on a range of complex samples [88–90]. Correspondingly, we also envisage that continued software development will enhance the assignment and quantitation of glycopeptide data, analysis of glycan IM results and interpretation of sophisticated intact MS spectra.…”

Section: Five-year Viewmentioning

confidence: 94%

Glycosylation profiling to evaluate glycoprotein immunogens against HIV-1

Behrens

Struwe

Crispin

2017

Expert Review of Proteomics

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Introduction Much of the efforts to develop a vaccine against the human immunodeficiency virus (HIV) have focused on the design of recombinant mimics of the viral attachment glycoprotein (Env). The leading immunogens exhibit native-like antigenic properties and are being investigated for their ability to induce broadly neutralizing antibodies (bNAbs). Understanding the relative abundance of glycans at particular glycosylation sites on these immunogens is important as most bNAbs have evolved to recognize or evade the dense coat of glycans that masks much of the protein surface. Understanding the glycan structures on candidate immunogens enables triaging between native-like conformations and immunogens lacking key structural features as steric constraints limit glycan processing. The sensitivity of the processing state of a particular glycan to its structural environment has led to the need for quantitative glycan profiling and site-specific analysis to probe the structural integrity of immunogens. Areas covered We review analytical methodologies for HIV immunogen evaluation and discuss how these studies have led to a greater understanding of the structural constraints that control the glycosylation state of the HIV attachment and fusion spike. Expert commentary Total composition and site-specific glycosylation profiling are emerging as standard methods in the evaluation of Env-based immunogen candidates.

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Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity

Cited by 145 publications

References 55 publications

Native Mass Spectrometry: What is in the Name?

Native Mass Spectrometry: What is in the Name?

Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation

Glycosylation profiling to evaluate glycoprotein immunogens against HIV-1

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