Rp c18 pre 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%.

Spots of interest visible on the films were gently excised from compatible silver-stained gels and analyzed by liquid chromatography coupled to a mass spectrometer in the Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). Samples were concentrated and desalted on a RP-C18 pre-column (Waters), 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 long) in a nanoACQUITY UPLC system, using a 45 minute linear acetonitrile gradient. The column outlet was directly coupled to an Electrospray ionization (ESI) ion source of a Orbitrap Velos type mass spectrometer (Thermo Scientific, Waltham, USA), operating in a regime of a data-dependent MS to MS/MS switch with HCD-type peptide fragmentation. An electrospray voltage of 1.5 kV was used.