Mass Spectrometry Analysis of AML Immunopeptidomes

MHC-Class I and II immunopeptidomes were measured in parallel from primary AML tumor samples (1 X 10⁸ cells per MHC preparation) and AML cell lines (1 X 10⁹ cells per MHC preparation) as previously described (see S1 File) [8 (link),20 (link),21 ]. Isolated HLA peptides were reconstituted in 12 μl of 0.1% formic acid and analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) or a Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, USA). Peptides were separated by capillary reverse phase chromatography on 20–24 cm reversed phase columns (100 μm inner diameter, packed in-house with ReproSil-Pur C18-AQ 3.0 m resin (Dr. Maisch GmbH)) using two-step linear gradients with increasing acetonitrile as previously described (see S1 File) [8 (link),21 ]. All primary AML tumor samples were measured with the Orbitrap Elite mass spectrometer and analyzed three times with complementary acquisition methods. The two cell line specimens (OCI-AML3 and MV4-11) were analyzed with the Fusion Lumos tribrid mass spectrometer.