Beh130 c18

Analysis of tryptic peptides by LC–MS/MS was achieved by injection of the samples onto a nano-liquid chromatography system (nanoACQUITYy, Waters, Manchester, UK) coupled via ESI to a MS consisting of a quadrupole and an orbitrap mass analyzer (Orbitrap QExcactive, Thermo Scientific, Bremen, Germany). Peptides were separated on a UPLC column, (BEH 130 C18, Waters; 75 μm × 250 mm, 1.7 µm, 100 Ǻ; 200 nL/min) by a linear gradient from 2 to 30% acetonitrile/0.1% formic acid in 120 min. Mass spectra were measured in the positive ion mode. LC–MS/MS analysis was done on MS level over a m/z range from 400–1500.

MS analysis of peptides and DMB-labelled sugars was performed on a nanoACQUITY UPLC system (Waters) equipped with an analytical column (Waters, BEH130C18, 100 μm × 100 mm, 1.7-μm particle size) coupled online with an ESI Q-TOF (Q-TOF Ultima, Waters). DMB-labelled sugars dissolved in acetonitrile/methanol/H₂O (9:7:84, v/v) and peptides dissolved in 2% acetonitrile and 0.1% formic acid were separated by reverse-phase chromatography using acetonitrile as eluent. MS spectra were recorded in positive reflection mode and analytes were automatically subjected to fragmentation (MS/MS). Spectra were analysed using MassLynx V4.1 software (Waters). MS/MS protein spectra were automatically analysed using the program ProteinLynx Global Server (Version 2.1, Waters).

HDX-MS experiment was performed as reported^{43 (link)}. Nm23-H1 15 μM were incubated with 100 μM NMac1 at R.T. for 30 min in 5% acetonitrile (ACN) followed by incubated with 20-fold D₂O at 25 °C for the following periods of time: 10, 60, 300, and 1800 s. The deuterium labeling reaction was quenched by 2.5 mM tris (2-carboxyethyl) phosphine (TCEP), formic acid, pH 2.3. For protein digestion, 1 μg of porcine pepsin was added to each quenched protein sample and incubated on ice for 3 min before injection. Peptic peptides were desalted on C₁₈ trap column cartridge (Waters) and gradient eluted from 8% ACN to 40% ACN, 0.1% formic acid on 100 µm i.d.× 100 mm analytical column, 1.7 µm particle size, BEH130 C₁₈ (Waters) for 7 min. The trap, analytical column and all tubing were immersed in an ice bath to minimize deuterium back-exchange. Gradient chromatography was performed at a flow rate 0.5 μL/min and was sprayed on line to nanoAcquity^TM/ESI/MS (SYNAPT^TM HDMS^TM) (Waters). The extent of deuterium incorporation was monitored the increase in mass of the isotope distribution for each identified peptide, and calculated by Microsoft Excel and DynamX v3. The theoretical maximum deuterium incorporation value was calculated for each peptide based on the number of exchangeable amides. Each experiment was triplicated.

Recombinant Nm23-H1 (2 μg/μL) was diluted 10-fold with 99% D₂O for 30 sec, 1, 3, 5, 10, 30, and 60 min and maintained at 25 °C with 1 mM H₂O₂. The labeling reaction was quenched by 5 mM tris(2-carboxyethyl)phosphine hydrochloride, pH 2.3 (This is titrated with formic acids). For peptic digestion, porcine pepsin (1 mg/mL) was added to each quenched protein sample and incubated at 0 °C for 3 min before injection^{21 (link)}.
Deuterated peptic peptides were desalted on line prior to separation using trap column (ID 180 μm × 20 mm, Symmetry® C18) cartridge. Peptides were separated using a C18 reversed-phase 100 μm ID × 100 mm analytical column (1.7 μm particle size, BEH130 C18, Waters Co. USA) with integrated electrospray ionization PicoTipTM (±10 μm ID, New Objective, USA). The auto-sampler chamber was set at 5 °C. The trap, analytical column and all tubing were immersed in an ice bath to minimize deuterium back-exchange. Both mobile phase bottles were placed on ice and both mobile phases contained 0.1% FA. Gradient chromatography was performed at 600 nL/min flow rate and was sprayed on line to mass spectrometer (SYNAPT^TM HDMS^TM, Waters Co. USA). All mass spectral measurements were taken at: capillary voltage 2.5 kV, cone voltage 35 V, extraction cone voltage 4.0 V, source temperature 80 °C. TOF mode scan was performed in range of m/z 300–1500 with scan time of 1 sec.