Hb a0

In total, 112 anonymized plasma samples were collected from surplus material from patient samples at the Department of Clinical Biochemistry and Pharmacology, Odense University Hospital in the period between September 2018 and November 2018. The study was conducted as a quality study with anonymous blood samples, which according to the legislation under the Danish Health Authorities does not require approval from ethical committee. The study was approved by the Region of Southern Denmark (reference number 18/51099). Samples were stored at -80 o C until further analysis. Hp phenotype was determined by western blotting. The samples showed the following distribution Hp1-1 (n=17, 15.2%), Hp2-1 (n=53, 47.3%) and Hp2-2 (n=42, 37.5%).
Native purified Hp1-1 and Hp2-2 from human serum were purchased (#H0138; #H9762; Sigma-Aldrich). Recombinant Hp1-1 (rHp1-1, NP_001119574.1) and recombinant Hpr (rHpr, AAA88081.1) were expressed using the ExpiCHO-S expression system (#A29133; ThermoFisher Scientific) according to manufacturer's instructions. rHp1-1 was expressed with a C-terminal His-Tag and was purified using a Ni-NTA Superflow column (#30430, Qiagen). On the other hand, rHpr was purified using Hb A0 (#H0267; Sigma-Aldrich) affinity chromatography.

Trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (α-CHCA) phosphorous red and TLC sheets were purchased from Sigma Aldrich (Poole, UK). Acetonitrile, (ACN) was purchased from Fisher Scientific (Loughborough, UK). Lyophilised sequencing grade modified Trypsin was obtained from Promega in 20 μg vials (Southampton, UK). RapiGest was obtained in 1 mg vials from Waters (Waters Corp, Manchester, UK). Double sided conductive tape was purchased from TAAB (Aldemastor, UK), Intellislides (glass slides) were made available by Bruker Daltonik (GmbH, Bremen, Germany). Indium tin oxide (ITO) slides were purchased from Sigma Aldrich (Poole, UK). Six Haemoglobin variants were investigated by MALDI MS, MS/MS and MSI namely, HbC, HbD-Iran, HbD-Punjab/Los Angeles, HbE, HbJ Baltimore and HbS. HbA 0 ("normal" Hb) was also included as a reference. Samples were available as standards (HbA 0 , and HbS were purchased from Sigma Aldrich (Dorset, UK) and a mixed Hb AFSC standard was purchased from Sebia (Lisses, France), as they were not commercially available individually, or as patients' blood. Patients' blood was provided by Sheffield Teaching Hospitals under the IRAS and the Sheffield Hallam University Ethics approvals 160418 and ER6558932 respectively. These samples were analysed using three types of MALDI Mass Spectrometers.