Massprep micro desalting column

1 μg of protein was loaded onto a MassPREP micro desalting column (Waters) and washed for 5 min with 10% (vol/vol) acetonitrile/0.1% formic acid. Following a 1-min gradient to 85% (vol/vol) acetonitrile/0.1% formic acid, the protein was eluted into a Xevo G2-XS QToF (Waters) using electrospray ionization for molecular mass measurement.

To generate CoA or dPCoA complexes with Aurora A, 10 μM WT Aurora A was incubated with 100 μM CoA or dpCoA for 15 min at room temperature. To evaluate the interaction, intact complexes were desalted using a C4 desalting trap (Waters MassPREP™ Micro desalting column, 2.1 × 5 mm, 20 μm particle size, 1000 Å pore size). Aurora A was eluted with 50 % (v/v) MeCN, 0.1 % (v/v) formic acid. Intact mass analysis was performed using a Waters nano ACQUITY Ultra Performance liquid chromatography (UPLC) system coupled to a Waters SYNAPT G2, as described [60 ]. Samples were eluted from a C4 trap column at a flow rate of 10 μL/min using three repeated 0–100 % acetonitrile gradients. Data was collected between 400 and 3500 m/z and processed using MaxEnt1 (maximum entropy software, Waters Corporation).

Urine samples were diluted in formic acid (0.1% (v/v) in HPLC grade water) to a protein concentration of approximately 5 pmol/μl. The samples were injected onto a C4 desalting trap (Waters MassPREP™ Micro desalting column, 2.1 × 5 mm, 20 μm particle size, 1000 Å pore size) (Waters, Manchester, UK) that was fitted on a Waters nano ACQUITY Ultra Performance liquid chromatography^® (UPLC^®) system. The chromatography system was coupled to a Waters SYNAPT™ G1 QTof mass spectrometer fitted with an electrospray source. Protein was eluted over a 10 min acetonitrile (ACN) gradient (5–95% (v/v)) at 40 μl/min. Data were collected between 500–3500 m/z. The data were processed using maximum entropy deconvolution (MAX ENT 1, Mass Lynx version 4.1, Waters) at 0.5 Da/channel over a mass range of 8500–10000 Da. The mass spectrometer was calibrated externally with horse heart myoglobin (1 pmol/μl, Sigma).

Positive ion electrospray ionization (ESI) mass spectra were acquired on a Waters Synapt G1 quadrupole time-of-flight mass spectrometer equipped with a megaflow ion source. The ion source settings were: capillary voltage 3.5 kV, sampling cone voltage 26 V, extraction cone 2.6 V, ion source block temperature 70°C, desolvation gas (N₂) flow 500 L/h (150°C). The instrument was calibrated using apomyoglobin. Mass spectra were acquired for the mass range m/z 300–2000 with a detector (MCP) voltage of 1700 V to increase the signal-to-noise ratio for multiply charged ions. MS spectra were processed using Masslynx software (v. 4.1) and spectrum deconvolution was carried out with the maximum entropy algorithm (MaxEnt 1) included in this software. The protein samples were desalted by reversed-phase chromatography using a Waters MassPREP Micro Desalting Column. Gradient and desalting flows were provided by a Waters nanoACQUITY UPLC pump and a Dionex Ultimate 3000 HPLC pump. Desalting was carried out with 0.23% formic acid (v/v) (Solvent A). Elution from the colum was carried out with a short acetonitrile gradient.