Oasis hlb μelution

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).

Samples were frozen and kept at −80 °C until cell lysis. Cell lysis was performed using SDC lysis buffer (1% sodium deoxycholate (SDC), 10 mm tris(2‐carboxyethyl) phosphinehydrochloride (TCEP), 40 mm chloroacetamide (CAA) and 100 mm TRIS, pH 8.0 supplemented with phosphatase inhibitors (PhosSTOP; Roche) and protease inhibitors (complete mini EDTA‐free; Roche) as described previously [14 (link)]. Proteins were digested using a two‐step digestion. First, proteins were digested for 2 h using LysC (Wako Chemicals Europe GmbH, Neuss, Germany) at 37 °C (enzyme to protein ratio 1 : 75). Next, trypsin (Sigma‐Aldrich) was added in an enzyme to protein ratio 1 : 50 and digestion was performed overnight at 37 °C. Next, samples were acidified, and desalted using Oasis HLB μElution (Waters, Etten‐Leur, The Netherlands). Samples were aliquoted for full proteome analysis (40 μg), in‐house build library (30 μg), phosphopeptide enrichment (200 μg). Phosphopeptides were enriched using the AssayMAP Bravo Platform (Agilent Technologies) using Fe(III)‐NTA cartridges (Agilent Technologies) as described previously [14 (link)]. Samples were dried and stored at −20 °C until LC–MS analysis.