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Mass spectrometry combined with strategic enzymatic digestion, selective derivatization and ultraviolet photodissociation for the identification and characterization of Immunoglobulin G antibodies
No full textImmunoglobulin G (IgG) antibodies represent important analytical targets both for their therapeutic properties and for their critical role in the adaptive immune response. While much of the primary structure is conserved across the IgG class, subtle changes in amino acid sequence and the presence or absence of post-translational modifications can have a profound effect on the function and therapeutic potential of a given antibody. As such, there remains a high demand for versatile analytical tools capable of both identification and complete structural characterization of IgGs. The work presented in this dissertation largely focuses on the development of mass spectrometry-based methods for the improved analysis of antibodies. This was accomplished using strategic enzymatic Brodbeltselectivity for regions of particular diagnostic value or to facilitate comprehensive structural characterization. A method based on chromophore-mediated 351 nm UVPD was developed as a means to streamline the identification of antibodies in mixtures by enhancing selectively for the third complementarity determining region of the IgG heavy chain (CDR-H3). The hypervariable sequences within this region serve as the primary determinant of antigen binding specificity and thus provide a molecular signature by which to differentiate unique antibodies. To accomplish this, a highly conserved cysteine residue located in the framework preceding the CDR-H3 region was exploited for selective tagging with an Alexa Fluor 350 (AF350) thiol-selective maleimide. This site-specific tagging combined with strategic enzymatic digestion and 351 nm UVPD allowed selective dissociation of only AF350-labeled peptides for facile discrimination of CDR-H3 sequences within a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) based workflow. Two variations of middle-down mass spectrometry based on either restricted Lys-C proteolysis or hinge-selective IdeS digestion combined with 193 nm UVPD were used for the characterization of monoclonal antibodies. Both strategies yielded considerably greater diagnostic sequence information when benchmarked against conventional collision- and electron-based activation methods. The Lys-C proteolysis method was found to have considerable implications for the analysis of serological antibody repertoires owing to its facile implementation into high-throughput proteomic workflows and ability to unambiguously differentiate unique CDR-H3 sequences. The development and implementation of a front-end dual spray reactor for high-throughput ion/ion-mediated bioconjugation is demonstrated for the enhanced structural characterization of unmodified and post-translationally modified peptide cations by 193 nm UVPD and CID. The ability to generate ion/ion complexes in real-time followed by efficient covalent conversion allowed integration of the dual spray reactor into a high-throughput LC-MS [superscript n] workflow for rapid derivatization of peptide mixtures.Chemistr
Characterization of Therapeutic Monoclonal Antibodies at the Subunit-Level using Middle-Down 193 nm Ultraviolet Photodissociation
No full textCharacterization of Therapeutic Monoclonal Antibodies at the Subunit-Level using Middle-Down 193 nm Ultraviolet Photodissociation
No full textMonoclonal
antibodies (mAbs) are a rapidly advancing class of therapeutic
glycoproteins that possess wide clinical utility owing to their biocompatibility,
high antigen specificity, and targeted immune stimulation. These therapeutic
properties depend greatly on the composition of the immunoglobulin
G (IgG) structure, both in terms of primary sequence and post-translational
modifications (PTMs); however, large-scale production in cell culture
often results in heterogeneous mixtures that can profoundly affect
clinical safety and efficacy. This places a high demand on analytical
methods that afford comprehensive structural characterization of mAbs
to ensure their stringent quality control. Here we report the use
of targeted middle-down 193 nm ultraviolet photodissociation (UVPD)
to provide detailed primary sequence analysis and PTM site localization
of therapeutic monoclonal antibody subunits (∼25 kDa) generated
upon digestion with recombinant immunoglobulin G-degrading enzyme
of Streptococcus pyogenes (IdeS) followed by chemical
reduction. Under optimal conditions, targeted UVPD resulted in approximately
60% overall coverage of the IgG sequence, in addition to unambiguous
glycosylation site localization and extensive coverage of the antigen-binding
complementarity determining regions (CDRs) in a single LC-MS/MS experiment.
Combining UVPD and ETD data afforded even deeper sequencing and greater
overall characterization of IgG subunits. Overall, this targeted UVPD
approach represents a promising new strategy for the comprehensive
characterization of antibody-based therapeutics
High-Throughput Bioconjugation for Enhanced 193 nm Photodissociation via Droplet Phase Initiated Ion/Ion Chemistry Using a Front-End Dual Spray Reactor
No full textFast online chemical derivatization
of peptides with an aromatic
label for enhanced 193 nm ultraviolet photodissociation (UVPD) is
demonstrated using a dual electrospray reactor implemented on the
front-end of a linear ion trap (LIT) mass spectrometer. The reactor
facilitates the intersection of protonated peptides with a second
population of chromogenic 4-formyl-1,3-benzenedisulfonic acid (FBDSA)
anions to promote real-time formation of ion/ion complexes at atmospheric
pressure. Subsequent collisional activation of the ion/ion intermediate
results in Schiff base formation generated via reaction between a
primary amine in the peptide cation and the aldehyde moiety of the
FBDSA anion. Utilizing 193 nm UVPD as the subsequent activation step
in the MS<sup>3</sup> workflow results in acquisition of greater primary
sequence information relative to conventional collision induced dissociation
(CID). Furthermore, Schiff-base-modified peptides exhibit on average
a 20% increase in UVPD efficiency compared to their unmodified counterparts.
Due to the efficiency of covalent labeling achieved with the dual
spray reactor, we demonstrate that this strategy can be integrated
into a high-throughput LC-MS<sup><i>n</i></sup> workflow
for rapid derivatization of peptide mixtures
Selective 351 nm Photodissociation of Cysteine-Containing Peptides for Discrimination of Antigen-Binding Regions of IgG Fragments in Bottom-Up Liquid Chromatography–Tandem Mass Spectrometry Workflows
No full textDespite tremendous inroads in the
development of more sensitive
liquid chromatography–tandem mass spectrometry (LC–MS/MS)
strategies for mass spectrometry-based proteomics, there remains a
significant need for enhancing the selectivity of MS/MS-based workflows
for streamlined analysis of complex biological mixtures. Here, a novel
LC–MS/MS platform based on 351 nm ultraviolet photodissociation
(UVPD) is presented for the selective analysis of cysteine–peptide
subsets in complex protein digests. Cysteine-selective UVPD is mediated
through the site-specific conjugation of reduced cysteine residues
with a 351 nm active chromogenic Alexa Fluor 350 (AF350) maleimide
tag. Only peptides containing the AF350 chromophore undergo photodissociation
into extensive arrays of b- and y-type fragment ions, thus providing a facile means for differentiating
cysteine–peptide targets from convoluting peptide backgrounds.
With the use of this approach in addition to strategic proteolysis,
the selective analysis of diagnostic heavy-chain complementarity determining
regions (CDRs) of single-chain antibody (scAb) fragments is demonstrated
Modulation of Phosphopeptide Fragmentation via Dual Spray Ion/Ion Reactions Using a Sulfonate-Incorporating Reagent
No full textThe
labile nature of phosphoryl groups has presented a long-standing challenge
for the characterization of protein phosphorylation via conventional
mass spectrometry-based bottom-up proteomics methods. Collision-induced
dissociation (CID) causes preferential cleavage of the phospho-ester
bond of peptides, particularly under conditions of low proton mobility,
and results in the suppression of sequence-informative fragmentation
that often prohibits phosphosite determination. In the present study,
the fragmentation patterns of phosphopeptides are improved through
ion/ion-mediated peptide derivatization with 4-formyl-1,3-benezenedisulfonic
acid (FBDSA) anions using a dual spray reactor. This approach exploits
the strong electrostatic interactions between the sulfonate moieties
of FBDSA and basic sites to facilitate gas-phase bioconjugation and
to reduce charge sequestration and increase the yield of phosphate-retaining
sequence ions upon CID. Moreover, comparative CID fragmentation analysis
between unmodified phosphopeptides and those modified online with
FBDSA or in solution via carbamylation and 4-sulfophenyl isothiocyanate
(SPITC) provided evidence for sulfonate interference with charge-directed
mechanisms that result in preferential phosphate elimination. Our
results indicate the prominence of charge-directed neighboring group
participation reactions involved in phosphate neutral loss, and the
implementation of ion/ion reactions in a dual spray reactor setup
provides a means to disrupt the interactions by competing hydrogen-bonding
interactions between sulfonate groups and the side chains of basic
residues
Middle-Down 193-nm Ultraviolet Photodissociation for Unambiguous Antibody Identification and its Implications for Immunoproteomic Analysis
No full textChemical Composition and Antifungal Activity of Lavender (Lavandula stoechas) Oil
No full textThe essential oil of Lavandula stoechas was examined by GC and GC-MS. Discs (5 mmi.d.) of the tested fungi (Alternaria alternata, Fusarium oxysporum and Botritys cinerea) were inoculated separately onto each assay plate and incubated at 25 degrees C for 7 days. The oil yield of dried parts (v/dw) obtained by hydro distillation was 2.9%. Thirty-two compounds representing 98.3% of the essential oil were determined. Linalool (49.9%), linalyl acetate (14.4%), lavandulyl acetate (5.7%), alpha-terpineol (5.6%), terpinene-4-ol (5.1%), lavandulol (3.7%), (E)-beta-ocimene (2.6%) and (Z)-beta-ocimene (2.4%) were identified as the main constituents of the oil. In addition, both doses of the lavender oil showed varying levels of inhibitory effects on the mycelial growth of tested fungi used in the experiment. The results demonstrated the strongest effect on B.cinerea, followed by A.alternata and F.oxysporum. The inhibitory effect is probably dependent on the concentration of essential oils.International Scientific Partnership Program ISPP at King Saud University [0015]The authors extend their appreciation to the International Scientific Partnership Program ISPP at King Saud University for funding this research work through ISPP# 0015. Author would like to thank Mrs Svetlana Bajic-Raymond from Cotham School, England, for valuable comments and suggesting the text corrections
The GCN2-ATF4 Signaling Pathway Induces 4E-BP to Bias Translation and Boost Antimicrobial Peptide Synthesis in Response to Bacterial Infection
No full textUVnovo: A <i>de Novo</i> Sequencing Algorithm Using Single Series of Fragment Ions via Chromophore Tagging and 351 nm Ultraviolet Photodissociation Mass Spectrometry
No full textDe novo peptide
sequencing by mass spectrometry
represents an important strategy for characterizing novel peptides
and proteins, in which a peptide’s amino acid sequence is inferred
directly from the precursor peptide mass and tandem mass spectrum
(MS/MS or MS3) fragment ions, without comparison to a reference
proteome. This method is ideal for organisms or samples lacking a
complete or well-annotated reference sequence set. One of the major
barriers to de novo spectral interpretation arises
from confusion of N- and C-terminal ion series due to the symmetry
between b and y ion pairs created
by collisional activation methods (or c, z ions for electron-based activation methods). This is known
as the “antisymmetric path problem” and leads to inverted
amino acid subsequences within a de novo reconstruction.
Here, we combine several key strategies for de novo peptide sequencing into a single high-throughput pipeline: high-efficiency
carbamylation blocks lysine side chains, and subsequent tryptic digestion
and N-terminal peptide derivatization with the ultraviolet chromophore
AMCA yield peptides susceptible to 351 nm ultraviolet photodissociation
(UVPD). UVPD-MS/MS of the AMCA-modified peptides then predominantly
produces y ions in the MS/MS spectra, specifically
addressing the antisymmetric path problem. Finally, the program UVnovo
applies a random forest algorithm to automatically learn from and
then interpret UVPD mass spectra, passing results to a hidden Markov
model for de novo sequence prediction and scoring.
We show this combined strategy provides high-performance de
novo peptide sequencing, enabling the de novo sequencing of thousands of peptides from an Escherichia
coli lysate at high confidence
