Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples

Abstract

Hemoglobin (Hb) constitutes an important protein in clinical diagnosticsboth in humans and animals. Among the high number of sequence variants, some can cause severe diseases. Moreover, chemical modifications such as glycation and carbamylation serve as important biomarkers for conditions such as diabetes and kidney diseases. In clinical routine analysis of glycated Hb, sequence variants or other Hb proteoforms can cause interference, resulting in wrong quantification results. We present a versatile and flexible capillary zone electrophoresis-mass spectrometry screening method for Hb proteoforms including sequence variants and modified species extracted from dried blood spot (DBS) samples with virtually no sample preparation. High separation power was achieved by application of a 5-layers successive multiple ionic polymer layers-coated capillary, enabling separation of positional isomers of glycated α- and β-chains on the intact level. Quantification of glycated Hb was in good correlation with the results obtained in a clinical routine method. Identification and characterization of known and unknown proteoforms was performed by fragmentation of intact precursor ions. N-Terminal and lysine glycation could be identified on the α- and β-chain, respectively. The versatility of the method was demonstrated by application to dog and cat DBS samples. We discovered a putative new sequence variant of the β-chain in dog (T38 → A). The presented method enables separation, characterization, and quantification of intact proteoforms, including positional isomers of glycated species in a single run. Combined with the simple sample preparation, our method represents a valuable tool to be used for deeper characterization of clinical and veterinary samples.

Polyelectrolyte Multilayers in Capillary Electrophoresis

“…In addition to analyzing therapeutic compounds, SMIL coatings in CE may be used to screen for biomarkers which allows the detection of illnesses such as diabetes and kidney disease [63] . Indeed, Stolz et al.…”

Immunoaffinity Capillary Electrophoresis in the Era of Proteoforms, Liquid Biopsy and Preventive Medicine: A Potential Impact in the Diagnosis and Monitoring of Disease Progression

“…Proteomics should ideally be the driver for improving diagnostics and will serve as a primary method for detection of molecular changes indicative of disease states. Numerous laboratories have reported a plethora of assays using capillary electrophoresis in conventional format and in microfluidic format for detection and quantification of several proteoforms [254][255][256][257][258][259][260][261][262][263][264][265][266][267]. However, in order for proteoforms to reach their full clinical potential, it is necessary to be able to accurately detect and quantify near all proteoforms in biological fluids using advanced and sophisticated analytical instrumentation, such as IACE.…”

Characterisation of a new online nanoLC-CZE-MS platform and application for the glycosylation profiling of alpha-1-acid glycoprotein

“…PVA capillaries were prepared as described elsewhere [36]. SMIL-coated capillaries were prepared as previously described [37]. For this study, two different combinations of polycation and polyanion were used: PDADAMAC-PMA and DEAEDq-PMA.…”

“…As the high content of sialic acids on the protein results in a low pI (2.8-3.8 [28]), hence low mobility in CZE, we decided to adapt the CZE-MS separation system. By using a SMIL coating with a lower EOF (DEAEDq-PMA), resolution can be enhanced for slowly migrating species [37]. CZE-MS resulted in a broad peak of overlapping glycoforms (Fig.…”