Hemoglobin Digestion and Proteomic Analysis

Hemoglobin digests were reduced with 5 mM DTT for 20 min at RT and subsequently alkylated with iodoacetamide for 20 min at 37°C. The samples were measured using an LTQ Orbitrap Velos Pro system (Thermo Fisher Scientific) online coupled to an U3000 RSLCnano (Thermo Fisher Scientific) uPLC as described previously (Mohr et al., 2015 (link)), with the following modifications: For separation, a binary gradient consisting of solvent A (0.1% FA) and solvent B (86% ACN, 0.1% FA) was employed. After loading onto the precolumn, the sample was concentrated and washed in 5% B for 5 min. In a first elution step, the percentage of B was raised from 5 to 15% in 5 min, followed by an increase from 15 to 40% B in 30 min. The column was washed with 95% B for 4 min and re-equilibrated for subsequent analysis with 5% B for 19 min. For visualization, spectral data was exported from the datafile using XCalibur Qual Browser 2.2 (Thermo Fisher Scientific, Bremen, Germany). Database searches were performed using PEAKs X¹ (Zhang et al., 2012 (link)). For peptide identification, MS/MS spectra were correlated with the UniProt human reference proteome set². Carbamidomethylated cysteine was considered as a fixed modification along with oxidation (M) as a variable modification. False discovery rates were set on the peptide level to 1%.