Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques

Luca Fornelli, Kristina Srzentić, Romain Huguet, Christopher Mullen, Seema Sharma, Vlad Zabrouskov, Ryan T. Fellers, Kenneth R. Durbin, Philip D Compton, Neil L Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.

Original languageEnglish (US)
Pages (from-to)8421-8429
Number of pages9
JournalAnalytical Chemistry
Volume90
Issue number14
DOIs
StatePublished - Jul 17 2018

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Mass spectrometry
Chemical activation
Monoclonal Antibodies
Ions
Photodissociation
Liquid chromatography
Biosimilar Pharmaceuticals
Immunoglobulin Isotypes
Solid state lasers
Mass spectrometers
Deconvolution
Photons
Immunoglobulin G
Molecules
Electrons
Proteins
Experiments

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Fornelli, Luca ; Srzentić, Kristina ; Huguet, Romain ; Mullen, Christopher ; Sharma, Seema ; Zabrouskov, Vlad ; Fellers, Ryan T. ; Durbin, Kenneth R. ; Compton, Philip D ; Kelleher, Neil L. / Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques. In: Analytical Chemistry. 2018 ; Vol. 90, No. 14. pp. 8421-8429.
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abstract = "Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40{\%} for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90{\%} of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.",
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Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques. / Fornelli, Luca; Srzentić, Kristina; Huguet, Romain; Mullen, Christopher; Sharma, Seema; Zabrouskov, Vlad; Fellers, Ryan T.; Durbin, Kenneth R.; Compton, Philip D; Kelleher, Neil L.

In: Analytical Chemistry, Vol. 90, No. 14, 17.07.2018, p. 8421-8429.

Research output: Contribution to journalArticle

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AU - Fornelli, Luca

AU - Srzentić, Kristina

AU - Huguet, Romain

AU - Mullen, Christopher

AU - Sharma, Seema

AU - Zabrouskov, Vlad

AU - Fellers, Ryan T.

AU - Durbin, Kenneth R.

AU - Compton, Philip D

AU - Kelleher, Neil L

PY - 2018/7/17

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