Accurate Identification of Hemoglobin Variants By Top-Down Protein Analysis Using Capillary Electrophoresis-High-Resolution Mass Spectrometry

Introduction

Structural characterization of hemoglobin (Hb) variants, particularly the mutant forms of α- and β-subunits, is of significant value in the clinical diagnosis of hemoglobinopathy. The conventional methods for identification of Hb variants in clinical laboratories can be inadequate due to the lack of detailed structural characterization when it goes to the analysis of those Hb variants with similar sizes and charge states.

High-resolution mass spectrometry (HR-MS) has been a central technology for structural characterization of proteins. As a novel approach for HR-MS protein analysis, top-down workflow analyzes proteins in intact state without prior enzymatic digestion, and it is capable of identifying unique proteoforms. Capillary electrophoresis (CE), as a powerful separation technology for proteins, has demonstrated excellent separation efficiency in the analysis of intact Hb forms and Hb subunits. CE can be coupled with HR-MS to form a CE-HR-MS system. By this means the CE separation is able to enhance the analytical power of HR-MS to allow for superior analytical performance in Hb analysis. In addition, when neutral coating of capillary is employed to suppress electroosmotic flow, CE separates analytes only by their individual electrophoretic mobilities, which enhances separation efficiency by maximizing the electrophoretic difference between the analytes. Thus, we established a neutral-coating CE-HR-MS method for accurate identification of Hb variants with top-down protein analysis strategy.

Method

An Orbitrap Q-Exactive Plus mass spectrometer was coupled with an ECE-001 CE unit through an EMASS-II ion source. A PS1 neutral-coating capillary was used for CE separation. Samples of red blood cells were lysed in water and diluted in 10 mM ammonium formate buffer for analysis.

Results

In the neutral-coating CE, since denaturing conditions were used, intact Hb forms were dissociated in background electrolyte, and baseline separation of individual Hb subunits was observed. Hb subunits in ion electropherograms followed the order of α-, β-, δ-, γ₍₁₎-, γ2- subunit.

The identification of Hb variants using CE-HR-MS is a two-step process: (1) intact-protein analysis that preliminarily identifies Hb subunits by precursor ions, and (2) top-down analysis that characterizes the primary structures and confirms the identification of Hb subunits by fragments produced from particular precursor ions. In intact-protein analysis, multiple charge states of each Hb subunit were observed in the mass spectra. At each charge state of a Hb subunit, a cluster of isotopic MS peaks were observed, corresponding to the isotopes of the molecule. All the MS peaks in a mass spectrum can be deconvoluted and those resulted from one analyte can be merged to display a single MS peak at its accurate monoisotopic mass, which can be matched to the theoretical mass of a known Hb subunit or variant. In top-down protein analysis, fragments from a precursor ion were acquired after HCD fragmentation. The MS peaks can be deconvoluted and those resulted from one fragment can be merged to display a single MS peak at its monoisotopic mass. The monoisotopic masses of fragments can be used to characterize the primary structure of the analyte by matching them to the theoretical masses of possible fragments from the structure of a known Hb subunit.

The CE-HR-MS method was applied to the analysis of normal Hb forms as well as Hb variants from adults and neonates. The structures of Hb subunit variants β-S, β-C, β-E, β-Riyadh, β-Koln, β-New York, β-G-Accra, β-G-Siriraj, β-Khartoum, β-D-Punjab, β-D-Iran, β-Deer Lodge, α-Tarrant, γ1-Kuala Lumpur, and P-Nilotic β-δ hybrid have been successfully identified and characterized with >30% amino acid residue coverage and >95% matched fragment coverage. As an example, the CE-HR-MS result for the identification of a Hb γ1/γ1-Kuala Lumpur is shown, with intact-protein analysis in Figure 1A and top-down analysis in Figure 1B.

Conclusion

We have utilized the neutral-coating CE-HR-MS method for top-down structural characterization of hemoglobin variants. With the use of CE, only a simple dilute-and-shoot sample preparation procedure is required. Baseline separation of Hb subunits can be achieved to enhance HR-MS data quality. With these advantages, the CE-HR-MS method is compatible with clinical laboratories and can potentially be used in routine clinical testing.