B-248 Enhanced Detection of of Hemoglobin Variants in Clinical Research using Dried Blood Spot and HRAM MS
Authors: Song Y , Hassell K , Goucher E , Guo J , Correa T 
Published: October 2024

Abstract

Background
Hemoglobinopathies are one of the most prevalent genetic disorders, with more than 330,000 affected births occurring annually. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. However, there are challenges to differentiate isomers (i.e., between Hb C and Hb E), very similar masses, or new variants due to their lack of resolution. Mass spectrometry (MS) gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins. Here, we present the top-down analysis for enhanced detection of various hemoglobin variants using Orbitrap Exploris for clinical research.

Methods
Proteins were directly extracted from dried blood spots (DBS) and analyzed by Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option coupled to Vanquish HPLC system. A 3.2 mm disc was punched from the DBS cards to 96 well plate and 50 uL water was added to re-hydrate. For protein precipitation, 150 uL acetonitrile was added and incubated at -20 ⁰C for 15 minutes. After micro-centrifugation, 160 uL of supernatant was removed and 70 uL water was added to resuspend. For LC-MS analysis, 10 uL of supernatant was diluted 8 times with mobile phase A and transferred to another 96-well plate. LC separation was performed on MAbPac RP column 4 um, 1 x 100 mm, and data-dependent acquisition mode was operated.

Results
With direct protein extraction from DBS, the entire sample preparation time is less than 1 hour. The MAbPac column showed its capability of separating between different chains with minor overlap between normal beta and HbS beta chains. Data processing was performed by ProSightPD, resulting in ∼45% sequence coverage for both normal Hb and HbS beta chains. The mass difference between normal beta and Hb S beta chains is about 30 Da, which results in about 1.6 m/z difference at a charge state of 19. This mass difference can often be hindered by other proteins or interferences from the matrix if the mass analyzer doesn’t have sufficient resolving power. Here we successfully applied Orbitrap resolution at 120,000 to isolate these similar masses for accurate variants or subunits identification including normal alpha, normal beta, Hb S, and delta chains. The confirmation of the delta chain can be beneficial since its abundance is usually less than 0.5% in adults, which shows potential for detecting low abundant variants using this method. Also, the calibration curve was generated to demonstrate the method's feasibility which is not a typical quantitation method for hemoglobinopathies. For quantitation, three most abundant m/z values from 2 charge states were added to TraceFinder for peak area extraction and normal Hb beta chain was used as an alternative internal standard for data processing. The 3-day inter/intraday analysis generated great reproducibility with CV &lt; 10% and LOQ was determined to be 2.5 mg/mL with R2 values &gt;0.994.

Conclusions
Successful demonstration of a single-step protein extraction directly from DBS and top-down analysis of Hb variant for clinical research.