High-Throughput Analysis of Hemoglobin from Neonates Using Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Abstract

The risks associated with sickle cell and other untreated hemoglobinopathies have led to mandatory neonatal screenings in 41 of the 50 states in the US (1). Preliminary screenings are often performed by electrophoresis at alkaline pH on cellulose acetate, with follow-up analysis of abnormal samples by acid electrophoresis on citrate agar (2). Alternatively, isoelectric focusing (IEF) or HPLC can be used in the screenings (3), but other techniques offer advantages. In the past decade, new mass spectrometry (MS) approaches, including matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, have been applied to the study of proteins (4). MALDI-TOF MS offers a potent means of analyzing constituent chains of hemoglobin A (HbA) directly from samples as small as a single red blood cell (5). Mass accuracy and resolution are sufficient to determine the presence of point mutations within chains differing in mass by as little as ∼20 Da. This ability allows detection of the β6Glu→Val variant (∼30 Da lower in molecular mass) during direct analysis of the intact chains. Moreover, the site of mutation can be determined by combining MALDI-TOF MS with specific enzymatic digestion, i.e., “mass mapping”. Thus, MALDI-TOF MS has the potential to be used in place of conventional approaches (6) for HbAS/HbS screening in the general population. To date, however, the mass spectrometric approaches have not been taken to the high-throughput mode necessary for efficient population screening, nor have they been applied to neonatal blood samples, which contain roughly equal portions of HbA and HbF. We report here progress in the development of a high-throughput mass spectrometric screening approach capable of screening the hemoglobin of neonates at rates of ∼100 samples/h. In a blind analysis, 96 neonate blood samples were prepared for both intact protein molecular mass determination and mass mapping using parallel robotics (96-well format) and analyzed …