Qual browser

For compounds discussed, the area under the curve (AUC) of the compound of interest was manually obtained through integration in Thermo FreeStyle and Qual Browser. Normalization to wild type (N2) was achieved by dividing the AUC of the compound of interest by the AUC of ascr#3, as detected in the exo-metabolome in ESI- ionization. The P values for metabolomics data were calculated via unpaired t tests with Welch correction.

The reaction mixture of in vitro methyltransferase assay was subjected to SDS-PAGE, and the bands on the gel were visualized by SimplyBlue™ SafeStain (Thermo Fisher Scientific). The bands corresponding to SUV39H2 were excised from the gel, and digested with sequencing grade TPCK-trypsin (Worthington Biochemical, Lakewood, NJ) and TPCK-chymotrypsin (Worthington Biochemical) in 30 μL of digestion buffer (10 mM Tris-HCl, 0.05% decyl glucoside, pH 8.0) at 37°C for 12 hours. The digest mixture was separated using a nanoflow LC (Easy nLC, Thermo Fisher Scientific) on an NTCC analytical column (C18, Φ0.075 × 100 mm, 3 μm, Nikkyo Technos, Tokyo, Japan) with a linear gradient of 35% buffer B (100% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min over 10 min, and subjected on-line to a Q-Exactive mass spectrometer (Thermo Fisher Scientific) with a nanospray ion source using data dependent TOP10 method. The MS/MS spectra were searched against the in-house database using local MASCOT server (version 2.5; Matrix Sciences, London, UK). The quantitative analysis using Qual Browser (version2.2; Thermo Fisher Scientific) was performed as described previously [46 ]. The coverage ratio of this LC-MS/MS analysis was 90.6% (Supplementary Figure S2).

The reaction mixture of in vitro methyltransferase assay was subjected to SDS-PAGE, and the bands on the gel were visualized by SimplyBlue^TM SafeStain (Life Technologies, Carlsbad, CA). The bands corresponding to LSD1 were excised from the gel, and digested with sequencing grade TPCK-trypsin (Worthington Biochemical, Lakewood, NJ) in 30 μL of digestion buffer (10 mM Tris-HCl, 0.05% decyl glucoside, pH 8.0) at 37°C for 12 h. The digest mixture was separated using a nanoflow LC (Easy nLC, Thermo Fisher Scientific, Waltham, MA) on an NTCC analytical column (C18, Φ0.075 × 100 mm, 3 μm, Nikkyo Technos, Tokyo, Japan) with a linear gradient of 35% buffer B (100% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min over 10 min, and subjected on-line to a Q-Exactive mass spectrometer (Thermo Fisher Scientific) with a nanospray ion source using data dependent TOP10 method. The MS/MS spectra were searched against the in-house database using local MASCOT server (version 2.3; Matrix Sciences, London, United Kingdom). The quantitative analysis using Qual Browser (version2.2; Thermo Fisher Scientific) was performed as described previously [7 ].

A low pH (pH 6.0) non-reduced lysyl endoproteinase Lys-C mapping technique was applied to mAb and ADC samples. The digested samples were acidified with TFA then analyzed by LC/MS on a Thermo LTQ Orbitrap XL coupled to an The Agilent 1260 HPLC. Peptides were separated over an XBridge, BEH130 C18 column (3.5 μm particle sizes, 2.1 mm × 150 mm, Waters PN: 186,003,023) maintained at 60 °C with a flow rate of 0.2 mL/min. Mobile phases A and B consisted of 0.05% TFA in water and 0.05% TFA in acetonitrile, respectively. Peptides were eluted from the column using a gradient designed to recover the expected non-reduced peptides and hydrophobic: 3.0–25.0% B in 22 min, 25.0–34.0% B in 9 min, and 34.0–71.0% B in 37 min. The mass spectrometer utilized internal lock mass ion of hexakis(1H,1H,3H-perfluoropropoxy)phosphazene at m/z 922.009798 for [M + H +]1 + via dynamic calibration. Theoretical molecular masses for peptides were calculated using Bruker Sequence Editor. The observed multiply-charged peptides were converted to neutral molecular masses using Xtract, a software component of Thermo Qualbrowser. Neutral, monoisotopic masses were reported for all peptides observed in the LC/MS data and UV absorbance was monitored at 214 nm.

LC-MS analysis was performed using an ESI-Orbitrap mass spectrometer (Exactive, Thermo Fisher Scientific, Waltham, MA, USA) coupled to a 1200 series HPLC (Chromolith^® Performance RP-18e 100–4.6 mm, Agilent Technologies, Santa Clara, CA, USA). A 20 min linear gradient from 100% water (+0.05% TFA) to 100% acetonitrile (+0.05% TFA) was used. The mass range was between 100 and 400 m/z. For MS peak integration, the software implemented in the mass analyzer was used (Qual Browser, Thermo Scientific, Waltham, MA, USA).