Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry

Ren, Da; Pipes, Gary D.; Hambly, David M.; Bondarenko, Pavel V.; Treuheit, Michael J.; Gadgil, Himanshu S.

doi:10.1016/j.ab.2008.09.026

Cited by 35 publications

(28 citation statements)

References 27 publications

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“…The LTQ-Orbitrap was used for top-down analysis of intact monoclonal IgG antibodies [27] and their light and heavy chains [18] during direct infusion as well as on-line with HPLC [3]. In addition, top-down (tandem mass spectroscopy, MS/MS) analysis of antibody chains was performed using a q-TOF mass spectrometer during direct infusion [28], and on-line with HPLC using in-source N-terminal fragmentation on an orthogonal-TOF instrument [29]. During production and formulation of a mAb, it is important to separate and quantify all isoforms of therapeutic antibody with different PTMs to further identify the sites of the modifications and their impact on potency, stability, and other critical attributes of the therapeutic molecules.…”

mentioning

confidence: 99%

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

Bondarenko

Second

Zabrouskov

et al. 2009

J. Am. Soc. Mass Spectrom.

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142

129

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Mass and top-down analyses of 150-kDa monoclonal immunoglobulin gamma (IgG) antibodies were performed on an Orbitrap analyzer. Three different sample delivery methods were tested including (1) infusion of an off-line desalted IgG sample using nano-electrospray; (2) on-line desalting followed by a step elution with a high percentage of organic solvent; and (3) reversed-phase HPLC separation and on-line mass and top-down analyses of disulfide isoforms of an IgG2 antibody. The accuracy of mass measurements of intact antibody was within Ϯ2 Da (15 ppm). The glycoforms of intact IgG antibodies separated by 162 Da were baseline resolved. In-source fragmentation of the intact antibodies produced mainly 115 residue fragments including N-terminal variable domains of heavy and light chains. The sequence coverage (the number of cleavages) was greatly increased after reduction of disulfide bonds and HPLC/MS/MS analysis of light and heavy chains using collisioninduced dissociation in the ion trap of the LTQ-Orbitrap. This is an attractive alternative to peptide mapping for characterization and monitoring of post-translational modifications attributed to minimal sample preparation, high speed of the mass/top-down analysis, and relatively minor method-induced sample modifications. [1,2]. This has resulted in a strong need for a highthroughput methods for analysis of different antibody drug candidates. Mass spectrometry has become one of the most powerful techniques for the structural characterization of monoclonal antibodies (mAbs) [3]. Traditionally, structural characterization of mAbs has been performed by a "bottom-up" approach after first digesting them to peptides [4 -6]. Unfortunately, enzymatic digestion is a laborious, time-consuming process and it often introduces artificial modifications, such as cyclization of N-terminal glutamine and deamidation [5,7]. Alternatively, protein molecular mass analysis is relatively fast, does not require lengthy sample preparation, and induces fewer, if any, modifications [8] compared with the peptide mapping [5,7]. Analysis of intact monoclonal IgG antibodies and their large domains has been reported for matrix-assisted laser desorption/ ionization (MALDI) and electrospray ionization (ESI) sources and almost all mass analyzers including MALDI-time of flight (TOF) [9 -11], ESI quadrupole (Q) [4,[12][13][14], ion trap [15], orthogonal TOF [8,11,16,17], and the LTQ-Orbitrap during direct infusion [18]. Although a mass change in a population of mAbs can be used to monitor post-translational modifications [8], it cannot reveal the site of modification. For that purpose, fragmentation of intact proteins, also known as "topdown" mass spectrometry, has been developed during the past decade [19 -25]

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mentioning

confidence: 99%

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

Bondarenko

Second

Zabrouskov

et al. 2009

J. Am. Soc. Mass Spectrom.

Self Cite

142

129

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“…3, upper right panel). Similar experiments have also been performed on TOF instruments, however, with less sequence information and at lower resolution (80). There are many other publications that tackle the challenging problems of therapeutics.…”

Section: Applications Of Intact Protein Msmentioning

confidence: 69%

Analysis of Intact Protein Isoforms by Mass Spectrometry

Tipton

Tran

Catherman

et al. 2011

Journal of Biological Chemistry

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The diverse proteome of an organism arises from such events as single nucleotide substitutions at the DNA level, different RNA processing, and dynamic enzymatic post-translational modifications. This minireview focuses on the measurement of intact proteins to describe the diversity found in proteomes. The field of biological mass spectrometry has steadily advanced, enabling improvements in the characterization of single proteins to proteins derived from cells or tissues. In this minireview, we discuss the basic technology for "top-down" intact protein analysis. Furthermore, examples of studies involved with the qualitative and quantitative analysis of full-length polypeptides are provided.

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“…The ionization mode was negative ion and the ESI capillary voltage was −3000 V and the cone voltage was 30 V. The collisionally activated dissociation (CAD) mass spectra were obtained using a Waters LCT Premiere ESI-TOF mass spectrometer according to the method described by Ren et al . [49] The ion guide 1 voltage was 65 V and the cone voltage was 30 V. The instrument was operated in negative ion mode.…”

Section: Methodsmentioning

confidence: 99%

Geographic Variability and Anti-Staphylococcal Activity of the Chrysophaentins and Their Synthetic Fragments

et al. 2012

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Drug-resistant Staphylococcus aureus is a continuing public health concern, both in the hospital and community settings. Antibacterial compounds that possess novel structural scaffolds and are effective against multiple S. aureus strains, including current drug-resistant ones, are needed. Previously, we have described the chrysophaentins, a family of bisdiarylbutene macrocycles from the chrysophyte alga Chrysophaeum taylori that inhibit the growth of S. aureus and methicillin-resistant S. aureus (MRSA). In this study we have analyzed the geographic variability of chrysophaentin production in C. taylori located at different sites on the island of St. John, U.S. Virgin Islands, and identified two new linear chrysophaentin analogs, E2 and E3. In addition, we have expanded the structure activity relationship through synthesis of fragments comprising conserved portions of the chrysophaentins, and determined the antimicrobial activity of natural chrysophaentins and their synthetic analogs against five diverse S. aureus strains. We find that the chrysophaentins show similar activity against all S. aureus strains, regardless of their drug sensitivity profiles. The synthetic chrysophaentin fragments indeed mimic the natural compounds in their spectrum of antibacterial activity, and therefore represent logical starting points for future medicinal chemistry studies of the natural products and their analogs.

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Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry

Cited by 35 publications

References 27 publications

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

Analysis of Intact Protein Isoforms by Mass Spectrometry

Geographic Variability and Anti-Staphylococcal Activity of the Chrysophaentins and Their Synthetic Fragments

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