High-Resolution and Tandem Mass Spectrometry – the Indispensable Tools of the XXI Century

Tsybin, Yury O.; Fornelli, Luca; Kozhinov, Anton N.; Vorobyev, Aleksey; Miladinović, Saša M.

doi:10.2533/chimia.2011.641

Cited by 7 publications

(8 citation statements)

References 46 publications

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“…Similar observations of specific ECD PIA distributions have been reported for larger systems, including proteins consisting of a bundle of 3 α-helix segments . Interestingly, Figures – also reveal very distinct preferential ETD fragmentation of intact IgGs . For example, Figure shows abundant z 86 , z 93 , z 97 , z 106 , and other product ions that are present in many charge states and clearly dominate the ETD PIA distribution.…”

Section: Discussionsupporting

confidence: 78%

“…The reasons for this failure in top-down mass spectrometry, as presented here, are the specific structural organization of IgGs, where the single glycosylation site is located in a middle of 50 kDa heavy chains and is protected by disulfide bonds. In addition to the overall MS/MS performance increase, top-down mass spectrometry performance for post-translational modification analysis could be further improved through increases in the signal-to-noise ratio of product ions through summation of data from more than 10 LC-MS/MS runs and extensive use of ion activation before and after ETD reaction. , Further increase in sequence coverage can be envisioned from an increased rate of MS/MS data acquisition, which would translate to a higher number of spectra per LC MS/MS run . Therefore, the use of CO 2 laser-based infrared multiphoton-induced ion activation should be considered …”

Section: Discussionmentioning

confidence: 99%

“…Presumably, these product ions indicate a structure-related facile backbone rupture within the IgG heavy chain. Indeed, these preferential cleavage sites are located between two highly structured, disulfide-protected domains of the heavy chain …”

Section: Discussionmentioning

confidence: 99%

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Structural Analysis of Intact Monoclonal Antibodies by Electron Transfer Dissociation Mass Spectrometry

et al. 2011

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Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

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Structural Analysis of Intact Monoclonal Antibodies by Electron Transfer Dissociation Mass Spectrometry

et al. 2011

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“…as realized in MS-based metabolomics and proteomics. [2] Nevertheless, the gap between the state-ofthe-art MS performance and the analytical demands imposed by the complexity of molecular systems of interest has not yet been bridged. [3,4] One of the limiting factors that delays further advancement in MS-based applications is the throughput of analysis in experiments with time constraints.…”

Section: Introductionmentioning

confidence: 99%

From High- to Super-resolution Mass Spectrometry

Tsybin

2014

Chimia

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High-resolution mass spectrometry (MS) is indispensable for the molecular-level analysis of biological and environmental samples with great intra- and inter-molecular complexity. Here, we summarize developments in Fourier transform mass spectrometry (FTMS), the flagship of high-resolution MS techniques, accomplished in our laboratory. Particularly, we describe the recent and envisioned progress in structural analysis of: i) isolated large proteins and their simple mixtures, with a focus on monoclonal antibodies, via top-down, middle-down, and extended bottom-up mass spectrometry; ii) complex protein mixtures and proteomes via extended bottom-up proteomics; and iii) crude oil fractions and similar complex molecular mixtures. Despite the unequivocal success in molecular structural analysis, the demonstrated results clearly indicate that the compromise between MS acquisition speed (throughput) and achievable resolution level inhibits further advances of MS applications in the areas related to life, environmental, and material sciences. To further advance beyond state-of-the-art FTMS capabilities in these areas, we present the technique of super-resolution mass spectrometry that has been pioneered by our laboratory.

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“…The commercially available FTMS platforms, ICR and Orbitrap, have already demonstrated the feasibility to obtain the isotopic fine structures of natural peptides and small proteins. ,,− Knowing the number of chemical elements in a peptide may significantly reduce the number of possible elemental compositions assigned to an ion with a given mass accuracy. − However, despite the advantages of having additional information about the elemental compositions of the peptides, the isotopic fine structure has not been routinely employed in the existing protein identification strategies. There are several factors that limit the routine implementation of isotopic fine structure mass spectrometry.…”

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confidence: 99%

On the Utility of Isotopic Fine Structure Mass Spectrometry in Protein Identification

et al. 2012

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Modern mass spectrometry (MS)-based protein identification and characterization relies upon accurate mass measurements of the (13)C isotopic distributions of the enzymatically produced peptides. Interestingly, obtaining peptide elemental composition information from its isotopic fine structure mass spectrum to increase the confidence in peptide and protein identification has not yet been developed into a bottom-up proteomics-grade analytical approach. Here, we discuss the possible utility and limitations of the isotopic fine structure MS for peptide and protein identification. First, we in silico identify the peptides from the E. coli tryptic digest and show the increased confidence in peptide identification by consideration of the isotopic fine structures of these peptides as a function of mass and abundance accuracies. In the following, we demonstrate that the state-of-the-art high magnetic field Fourier transform ion cyclotron resonance (FT-ICR) MS allows a routine acquisition of the isotopic fine structure information of a number of isobaric peptide pairs, including a pair of peptides originating from E. coli. Finally, we address the practical limitation of the isotopic fine structure MS implementation in the time-constraint experiments by applying an advanced signal processing technique, filter diagonalization method, to the experimental transients to overcome the resolution barrier set by the typically applied Fourier transformation. We thus demonstrate that the isotopic fine structures of peptides may indeed improve the peptide and possibly protein identification, can be produced in a routine experiment by the state-of-the-art high resolution mass spectrometers, and can be potentially obtained on a chromatographic time-scale of a typical bottom-up proteomics experiment. The latter one requires at least an order of magnitude increase in sensitivity of ion detection, which presumably can be realized using high-field Orbitrap FTMS and/or future generation of ultrahigh magnetic field FT-ICR MS equipped with harmonized ICR cells.

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High-Resolution and Tandem Mass Spectrometry – the Indispensable Tools of the XXI Century

Cited by 7 publications

References 46 publications

Structural Analysis of Intact Monoclonal Antibodies by Electron Transfer Dissociation Mass Spectrometry

Structural Analysis of Intact Monoclonal Antibodies by Electron Transfer Dissociation Mass Spectrometry

From High- to Super-resolution Mass Spectrometry

On the Utility of Isotopic Fine Structure Mass Spectrometry in Protein Identification

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