SDS-lysed patient and cell line samples were processed and digested according to the filter-aided sample preparation (FASP) method [23 (link),24 (link)]. All of the filter-processed samples used 20 μg of protein material. Peptides from both patient and cell line samples were cleaned up with the Oasis HLB μElution (Waters, Milford, MA, USA) protocol.4.4. Liquid Chromatography (LC)-MS Analysis.
Dried peptides were dissolved in 20 μL of 2% acetonitrile (ACN) and 0.5% formic acid (FA). Differently preserved THP-1 and Molm-13 samples were analyzed on an Orbitrap Elite mass spectrometer equipped with a nanospray Flex ion source coupled to an Ultimate 3000 Rapid Separation LC system (both from Thermo Scientific, Waltham, MA, USA). Approximately 0.5 μg peptides were pre-concentrated and separated, as previously described [5 (link)]. Patient samples without or with PBS wash(es) were analyzed on a Q Exactive HF Orbitrap mass spectrometer equipped with an Easy-Spray (Thermo Scientific) coupled to an Ultimate 3000 Rapid Separation LC system. Approximately 0.6 μg peptides were pre-concentrated on a 2 cm × 75 µm ID Acclaim PepMap 100 trapping column and separated on a 50 cm × 75 µm ID Easy-Spray PepMap RSLC analytical column (both from Thermo Scientific). Bound peptides were eluted within a 195 min run using a binary gradient with buffer A (0.1% FA in water) and buffer B (0.1% FA in ACN).
Dried peptides were dissolved in 20 μL of 2% acetonitrile (ACN) and 0.5% formic acid (FA). Differently preserved THP-1 and Molm-13 samples were analyzed on an Orbitrap Elite mass spectrometer equipped with a nanospray Flex ion source coupled to an Ultimate 3000 Rapid Separation LC system (both from Thermo Scientific, Waltham, MA, USA). Approximately 0.5 μg peptides were pre-concentrated and separated, as previously described [5 (link)]. Patient samples without or with PBS wash(es) were analyzed on a Q Exactive HF Orbitrap mass spectrometer equipped with an Easy-Spray (Thermo Scientific) coupled to an Ultimate 3000 Rapid Separation LC system. Approximately 0.6 μg peptides were pre-concentrated on a 2 cm × 75 µm ID Acclaim PepMap 100 trapping column and separated on a 50 cm × 75 µm ID Easy-Spray PepMap RSLC analytical column (both from Thermo Scientific). Bound peptides were eluted within a 195 min run using a binary gradient with buffer A (0.1% FA in water) and buffer B (0.1% FA in ACN).
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