MSACL 2022 Abstract

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. As a powerful separation technology for proteins, CE 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.

Capillary zone electrophoresis (CZE) is an ideal CE mode to couple with HR-MS analysis due to its simple electrolyte composition and high-resolution separation performance. Neutral coating-based CZE separates analytes only by their individual electrophoretic mobilities, which enhances separation efficiency by maximizing the electrophoretic difference between the analytes during the electrophoresis. Thus, we report a CZE-HR-MS method for top-down structural characterization of Hb variants.

Method

An Orbitrap Q-Exactive Plus mass spectrometer was coupled with an ECE-001 CE unit through an EMASS-II ion source. A PS1 neutrally coated capillary was used for CZE separation. The eletrolyte for CZE was 20% acetonitrile in water with 2% formic acid. CZE voltage was set at 30 kV, and ESI voltage was controlled at 2.2 kV.

Results

In the neutral coating-based CZE, since denaturing conditions were used, intact Hb forms were dissociated in BGE, and the separation of individual Hb subunits was observed. Hb subunits in ion electropherograms followed the order of α-, β-, γ(1)-, γ(2)-, δ-subunit.

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 monoisotopic mass, which can be used to identify the analyte by matching it to the theoretical mass of a known Hb subunit. In top-down protein analysis, a secondary mass spectrum of product ions was acquired after HCD fragmentation of the isotopic precursor ions in a small m/z window. 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 first-order structure of the analyte by matching them to the theoretical masses of possible fragments from the structure of a known Hb subunit.

The CZE-HR-MS method was applied to the analysis of normal Hb forms as well as Hb variants from adults and neonates. The structures of α-, β-, γ(1)-, γ(2)-, δ-, sickel-β-, C-β-, E-β-, Riyadh-β-subunits have been successfully identified and characterized with ~30% amino acid residue coverage and >90% fragment coverage.

Conclusion

We have utilized the neutral coating-based CZE-HR-MS method for top-down structural characterization of hemoglobin variants. With the use of CZE, 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 CZE-HR-MS method is compatible with clinical laboratories and can potentially be used in routine clinical testing.