Article pubs.acs.org/ac
Quantitative Mass Spectrometry Analysis of Intact Hemoglobin A2 by Precursor Ion Isolation and Detection Adelina E. Acosta-Martin,†,‡ Didia Coelho Graça,† Paola Antinori,† Lorella Clerici,‡ Ralf Hartmer,§ Markus Meyer,§ Denis Hochstrasser,†,‡ Kaveh Samii,∥ Pierre Lescuyer,†,‡ and Alexander Scherl*,†,‡ †
Department of Human Protein Sciences, Faculty of Medicine, Geneva University, Geneva, Switzerland Department of Genetic and Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland § Bruker Daltonics, Bremen, Germany ∥ Division of Hematology, Geneva University Hospital, Geneva, Switzerland ‡
ABSTRACT: Precise and accurate quantification of proteins is essential in clinical laboratories. Here, we present a mass spectrometry (MS)-based method for the quantification of intact proteins in an ion trap mass spectrometer. The developed method is based on the isolation and detection of precursor ions for the quantification of the corresponding signals. The method was applied for the quantification of hemoglobin (Hb) A2, a marker used for the diagnosis of a βthalassemia trait. The α and δ globin chains, corresponding to total Hb and HbA2, respectively, were isolated in the ion trap at specific charge states and ejected without activation. Areas of the corresponding isolated precursor ions were used to calculate the δ to α ratio. Three series of quantifications were performed on 7 different days. The standard curve fitted linearly (R2 = 0.9982) and allowed quantification of HbA2 over a concentration range from 3% to 18% of total Hb. Analytical imprecision ranged from 3.5% to 5.3%, which is enough to determine if the HbA2 level is below 3.5% or above 3.7%. In conclusion, our method reaches precision requirements that would be acceptable for the quantitative measurement of diagnostic proteins, such as HbA2, in clinical laboratories. (Table 1). Since each of these Hb types contains two α chains per tetramer, the amount of α chain in the sample will be
P
rotein assays in clinical laboratories have to meet specifications for imprecision and inaccuracy, allowing clinicians to make reliable decisions for patient care based on laboratory results. Desirable specifications for a given analyte can be calculated from intra- and interindividual biological variations.1 In this context, the complexity of most MS protocols developed for protein quantification represents a limitation for their use in clinical laboratories, mainly because of the resulting lack of robustness.2 Trypsin digestion, in particular, was shown to be a major source of analytical variability.3 In this paper, we describe a MS-based method for intact protein quantification in an ion trap mass spectrometer. The method applies a modified version of classical tandem MS, in which isolated ions are not activated but directly ejected and detected after isolation. By doing this, the obtained MS2 spectra, corresponding to isolated precursor ion signals, have an increased signal-to-noise ratio (S/N) compared to the one observed in MS1 spectra. Peak areas of isolated precursor ions are used for quantification. The evaluation of the method was performed on the quantification of hemoglobin (Hb)A2 in a whole blood sample. HbA2 quantification is performed in clinical laboratories for Hb disorder diagnosis and typically allows the identification of heterozygous carriers of β-thalassemia mutations (β-thalassemia trait).4 HbA2 measurement represents a particular challenge in terms of analytical goals.5 In normal adult blood, HbA (α2β2), HbA2 (α2δ2), and HbF (α2γ2) represent the total Hb content © 2013 American Chemical Society
Table 1. Typical Hemoglobin Concentration Values hemoglobin type
globin chain composition
healthy adult
β-thalassemia trait
HbA HbA2 HbF
2 α and 2 β chains 2 α and 2 δ chains 2 α and 2 γ chains
∼97%
