Exploring Oxidative Reactions in Hemoglobin Variants Using Mass Spectrometry: Lessons for Engineering Oxidatively Stable Oxygen Therapeutics

Abstract

Significance: Worldwide demand has driven the development of hemoglobin (Hb)-based oxygen carriers (HBOCs) as potential acellular oxygen therapeutics. HBOCs have the potential to provide an oxygen bridge to patients and minimize current problems associated with supply and storage of donated blood. However, to date, safety and efficacy issues have hampered the approval of viable HBOCs in the United States. These previous efforts have underscored the need for a better molecular understanding of toxicity to design safe and oxidatively stable HBOCs. Recent Advances: High-resolution accurate mass (HRAM) mass spectrometry (MS) has recently become a versatile tool in characterizing oxidative post-translational modifications that occur in Hb. When integrated with other analytical techniques, HRAM data have been invaluable in providing mechanistic insight into the extent of oxidative modification by quantifying oxidation in amino acids near the reactive heme or at specific "oxidative hotspots."

Critical issues: In addition to providing a deeper understanding of Hb oxidative toxicity, HRAM MS studies are currently being used toward developing suitable HBOCs using a "two-prong" strategy that involves (i) understanding the mechanism of Hb toxicity by evaluating mutant Hbs identified in patients with hemoglobinopathies and (ii) utilizing this information toward designing against (or for) these reactions in acellular oxygen therapeutics that will result in oxidatively stable protein.

Future directions: Future HRAM studies are aimed at fully characterizing engineered candidate HBOCs to determine the most oxidatively stable protein while retaining oxygen carrying function in vivo. Antioxid. Redox Signal. 26, 777-793.

Keywords: hemoglobin mutants; mass spectrometry; oxidation reactions.

FIG. 1.

Recombinant and biochemical strategies for the production of Hb-based oxygen carriers as blood substitutes are illustrated. (A) Tetrameric stabilization accomplished by joining two α/β heterodimers through intramolecular crosslinking between two α or two β subunits by either genetically engineering crosslinked recombinant Hb or a site-specific bifunctional crosslinker such as DBBF/DCLHb or NFPLP. (B) Effective molecular weight and surface area of Hb are increased by conjugating to polyethylene glycol (PEG-2000 or 5000). (C) Hb polymerization to create molecular weights significantly greater than native tetrameric (64 kDa) is produced when Hbs are reacted with polyfunctional crosslinking agents (e.g., glutaraldehyde, O-raffinose, O-adenosine). NFPLP, 2-nor-2-formyl pyridoxal-5′-phophate; DBBF, di-bromo bis-fumarate; DCLHb, diaspirin crosslinked Hb. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Graphic representation of a typical genetically engineered Hb (Optro (rHb 1.1 deoxy). This recombinantly expressed (in Escherichia coli) Hb-based oxygen carrier (Hb Presbyterian [βN108K], blue) is intramolecularly crosslinked via an αα glycine linker (red color). Highlighted (blue) ribbon represents the two linked amino (Val1 and Arg141) acids. Distances were measured and the theoretical model was generated using the PyMOL Molecular Graphics System distance measuring utility and mutagenesis wizard, respectively. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

Basic setup for a mass spectrometer and mass analyzers currently used with ESI-based mass spectrometers. (A) The basic setup for a mass spectrometer consists of an ion source, a mass analyzer that measures the mass-to-charge ratio (m/z) of gas-phase ions, and a detector that registers the number of ions at each m/z value. Most MS configurations are generally coupled online to a UPLC or HPLC system. In this illustration, ESI is shown where the ESI source located at the spray needle. (B) Commonly used mass spectrometer configurations (for protein/PTM identifications) with one or more of these analyzers are illustrated. (1) The ion-trap first captures ions (for a short time interval) and subjects them to either MS or fragmentation analysis (MS/MS). (2) The Q-TOF mass spectrometer typically involves ion selection via a quadrupole mass filter (Q) followed by fragmentation in a collisional cell (q2); the fragment ions then travel through the TOF analyzer at a rate proportional to their m/z ratio. (3) The FT-ICR analyzer captures ions under high vacuum in a high magnetic field; the ions orbit within the cyclotron at a frequency proportional to their m/z ratio. (4) The Q-Exactive mass spectrometer involves ion selection with a quadruple mass filter, ion capture in the Orbitrap analyzer followed by fragmentation in a HCD cell, and analysis in the Orbitrap. ESI, electrospray ionization; FT-ICR, Fourier transform ion cyclotron; HPLC, high-performance liquid chromatography; PTM, post-translational modification; TOF, time-of-flight; UPLC, ultra-performance liquid chromatography. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Schematic diagram of the bottom-up strategy (LC/MS/MS analysis). This strategy involves enzymatic digestion (top left corner) of intact proteins into peptides that are then analyzed by the mass spectrometer (top right corner). The resulting peptides are fragmented by CID (or HCD) (lower right corner) to generate MS/MS spectra that are then analyzed by database search algorithms (such as Mascot/SEQUEST) (lower left corner) for sequence information, in this case the Hb β gene sequence. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

Schematic diagram of label-free quantitative mass spectrometry strategy. Schematic diagram illustrating the basic workflow for quantitative mass spectrometry utilized toward characterizing Hb-based oxygen. Peptides are chromatographically separated, ionized via electrospray, and analyzed (top left corner) during a gradient run. The total ion chromatogram represented in the top right corner consists of thousands of MS (full scan) and MS/MS spectra that are first analyzed by Step 1 (bottom-up strategy) to identify oxidized Hb peptides followed by Step 2, a label-free method of quantifying the abundance of oxidized peptides relative to the unmodified peptide. To see this illustration in color and view labels, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Mass spectrometric characterization of Toms River's patient blood and the recombinant Hb γVal67Met counterpart. Displayed above the panels is the primary sequence of V67 M showing the tryptic peptide (highlighted in red) with position M67 (highlighted in blue). All charge states of this peptide sequence were the target of our quantitative proteomic approach. Fragmentation spectra of the singly charged +1 γV67 M (A) and γV67D tryptic peptides representing residues 67–76 from patient hemolysates are shown in (B). Both spectra show singly charged y and b fragment ions. (C) Isotopic profile of the singly charged γV67D tryptic peptide listed above. (D) Extracted ion chromatogram (XIC) of γV67D tryptic peptide (residues 67–76). XICs were generated from the most abundant monoisotopic peak represented in the isotopic profile of each targeted peptide. (E) Histogram representing the fold-change differences of V67D for experimental conditions 1–6 (1: CO ligand control, 2: control (air equilibrated buffer), 3: 1:1 H₂O₂, 4: 2.5:1 H₂O₂, 5: 5:1 H₂O₂, 6: Ferric/5:1 H₂O₂). These data indicate M to D conversion increases with increasing H₂O₂. (F) The isotopic profile represented by labeled isotopologous peptides after oxidation with H₂¹⁸O_2. The most abundant isotopic peak (M+H) of 1036 m/z represents the γV67D tryptic peptide with 2 ¹⁸O labeled carboxyl oxygen molecules. The 1034.56 m/z peak represents the γV67D tryptic peptide with a mixture of ¹⁸O/¹⁶O labeled carboxyl oxygen, and the lowest abundance 1032.56 m/z represents the unlabeled peptide (M + H). ¹⁸O monosubstituted and unlabeled γV67D tryptic peptide, respectively. This research was originally published in a study by Strader et al. (89) © the American Society for Biochemistry and Molecular Biology.

FIG. 7.

Structures of the active sites of Hb Toms River γV67 M, Hb Bristol-Alesha βV67 M, and Hb Evans αV62 M. (A) Electron density of γ MetE11 in HbCO Toms River 2Fo-Fc, refined electron density (4MQK). (B) Comparisons of MetE11 side-chain orientations in refined models for the CO forms of Hb γV67 M (4MQK), Hb βV67 M (4MQG), and Hb αV62 M (4MQC). (C) Oxidation of Hb βV67 M crystals (HbCO form, 4MQG; metHb form after 4 weeks, 4MQI). The electron density suggests the appearance of D at E11 position due to oxidation. The ligand electron density also suggests a ferryl complex with a single coordinated distal atom rather than aquomet complex with a spherical water O atom about 2 Å away from the central iron atom. (D) Oxidation of αV62 M crystals (HbCO form, 4MQC; metHb form after 7 weeks, 4MQH). There was no change in the electron density for the αE11 side chain in the oxidized Hb indicating no modification of the M62 residue. However, the coordination in the wild-type met-β active site again suggests a significant fraction of ferryl complex. This research was originally published in a study by Strader et al. (89) © the American Society for Biochemistry and Molecular Biology.

FIG. 8.

Isotopic profile and XIC of βCys93 tryptic peptide are illustrated. XICs were generated from the most abundant monoisotopic peaks of each isotopic profile. (A) Representative isotopic profile of the triply charged βC93 tryptic peptide GTFATLSELHCDKLHVDPENFR. (B) XIC of βC93 tryptic peptide (residues 83–104). (C) LC-MS/MS deconvoluted spectrum of βC93 tryptic peptide from HbS with DMPO adducts. The reaction solution of HbS with H₂O₂ in the presence of DMPO was digested with trypsin and analyzed by LC-MS/MS. The fragmentation spectra of the triply charged DMPO-labeled βC93 tryptic peptide GTFATLSELHCDKLHVDPENFR are shown. These spectra show singly charged y and b fragment ions. *Corresponds to y and b ions shifted in mass by 111 Da (from expected mass) confirming the location of the DMPO adducts to be Cys93. This research was originally published in a study by Kassa et al. (42) © the American Society for Biochemistry and Molecular Biology. XIC, extracted ion chromatogram.

FIG. 9.

Pseudoperoxidase catalytic of Hb. Spectral analysis H₂O₂ oxidation reactions: Absorbance spectra obtained at intervals of 2 min for 1 h on treatment of (A) HbA and (B) βK82D with 30:1 H₂O₂/heme equivalents. Note that while HbA undergoes the transition from ferrous to ferryl heme, spectral changes indicative of oxidative modification and some denaturation were evident, whereas in the case of Hb Providence (βK82D), H₂O₂ was consumed more efficiently as ferryl reverted back to ferric with little or no oxidative changes. (A, B) Reprinted with permission from Abraham et al. (1).