Beh130 c18 column

The protein spots were excised from the gel and analysed by liquid chromatography coupled to the mass spectrometer in the Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). Samples were subjected to a standard procedure of trypsin digestion during which proteins were reduced with 100 mM DTT (30 min at 56 °C), alkylated with 0.5 M iodoacetamide (45 min in a darkroom at room temperature), and digested overnight with 10 ng/µL trypsin (Promega, Madison, WI, USA) at 37 °C. The peptide mixtures were concentrated and desalted on an RP-C18 precolumn (Waters, Budapest, Hungary), and further peptide separation was achieved on a nano-Ultra Performance Liquid Chromatography (UPLC) RP-C18 column (Waters, BEH130 C18 column, 75 µm i.d., 250 mm length) of a nanoACQUITY UPLC system, using a 160 min gradient from 5 to 30% of acetonitrile. The column outlet was directly coupled to the electrospray ionization (ESI) ion source of the Orbitrap Elite type mass spectrometer (Thermo Scientific, Waltham, MA, USA), working in the regime of data-dependent MS to MS/MS switch with HCD type peptide fragmentation. An electrospray voltage of 2 kV was used. A blank run to ensure there was no cross contamination from previous samples preceded each analysis.

Proteomic analyses presented in this work were performed in the Laboratory of Mass Spectrometry at IBB PAS. Briefly, gel-excised protein samples were reduced with 100 mM DTT (30 minutes at 56°C) and alkylated with 0.5 M iodoacetamide (45 minutes in a darkroom at RT), followed by trypsin (10 ng µl⁻¹, Promega) overnight digestion at 37°C. Peptide mixtures were concentrated, desalted on a RP-C18 precolumn (Waters), and separated on a nano-Ultra Performance Liquid Chromatography (UPLC) RP-C18 column (Waters, BEH130 C18 column, 75 µm i.d., 250 mm long), using a 160 min gradient from 5 to 30% of acetonitrile. Measurements were performed with the Orbitrap Velos spectrophotometer (Thermo Fisher Scientific), working in the regime of data-dependent MS to MS/MS switch with HCD type peptide fragmentation. Identification of proteins was performed using the Mascot search engine (Matrix Science) with the probability-based algorithm. Data were searched with automatic decoy database and filtered to obtain a false discovery rate below 1%.

The proteins eluted from the THABS DNA affinity chromatography column were subjected to SDS-PAGE, and the gel was fixed in a solution containing acetic acid and methanol prior to staining in Colloidal Coomassie Brilliant Blue solution. The desired protein species was identified by alignment of the gel with an aliquot of the eluate blotted with anti-Thap1 and/or anti-V5 antibodies. The 32 kDa or 50 kDa Thap1 species was isolated, and the corresponding gel piece was excised and analyzed by mass spectrometry after trypsin digestion. The analysis was performed using reversed-phase LC over a Waters BEH130 C18 column (75 μm × 150 mm, 1.7 μm particle size) in a Waters NanoAcquity UPLC system (Waters, Milford, MA) interfaced to a Thermo LTQ-Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA).

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.

The sample was loaded onto the trap column (2 cm ×75 μm i.d., Symmetry C18), and then separated on a nanoACQUITY UPLC System (Waters, USA) equipped with a 25 cm ×75 μm i.d. BEH130 C18 column (Waters, USA) using a 5–35% buffer B (buffer A: 0.1% formic acid; buffer B: 0.1% formic acid in acetonitrile) gradient as the separation phase and a flow rate of 300 nl/min. The total running time was 120 min. The mass spectrometric data were collected on a high-resolution Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) operating in the data-dependent mode. Full MS resolution was set to 60,000 at 200 m/z and the mass range was set to 350–1600. dd-MS2 resolution was set to 15,000 at 200 m/z. Isolation width was set to 1.3 m/z. Normalized collision energy was set to 28%. The LC-MS/MS data were matched with the human SwissProt database using the Mascot search engine v.2.6.1 (Matrix Science, UK) with the following parameters: the mass tolerance of precursor peptide was set to 10 ppm, and the tolerance for MS/MS fragments was 0.02 Da.