Biobasic 18 column

Each cell pellet was lysed and it had protein digested with trypsin as detailed in the Supplementary Methods. Tryptic digest eluted in 0.1% formic acid (FA) in water was injected to an LTQ Velos dual-pressure ion trap mass spectrometer (Thermo Fisher Scientific) that was interfaced to a nano-electrospray ion source (spray voltage 1.6 kV, capillary temperature 250 °C), a Thermo Accela 600 pump, and an Accela autosampler LC. A BioBasic-18 column was used (dimension: 150.0 × 0.1 mm, particle size: 5 μm; Thermo Scientific). Solvents A and B in the mobile phase (flow rate: 150 μl min⁻¹) were 0.1% FA in water and 0.1% FA in acetonitrile, respectively. The chromatographic conditions and data acquisition method are described in the Supplementary Methods. LC-MS grade water and acetonitrile were from J. T. Baker. All other supplies and reagents were from Sigma-Aldrich, unless specified.

Peptides bound to A2.1 in the cell surface of either K562/A2.1 or K562/A2.1–S100-β were eluted by a brief incubation in acid citrate buffer (pH 3.3) and sequentially enriched using a 3 kDa cutoff Amicon Ultra Filter (MilliporeSigma, Burlington, MA, USA) and a Discovery DSC-18 trifunctional C18 silica resin column (MilliporeSigma). Peptide fractionation was done using a 150 mm × 2.1 mm BioBasic 18 column (Thermo Fisher Scientific), and peptide-containing fractions were stored at −80°C until analysis by mass spectrometry (MS).
MS was carried out in the Spectrometry Service [Instituto de Investigaciones Sanitarias (IDIS), Santiago de Compostela] on a matrix-assisted laser desorption/ionisation–time of flight mass spectrometry (MALDI-TOF) MS Analyzer (Thermo Fisher Scientific). The analysis was carried out with the 4000 Series Explorer software v.3.5 (Thermo Fisher Scientific) and Mascot v.2.1 (Matrix Science, Boston, MA, USA) to search against a National Center for Biotechnology Information nonredundant (NCBInr) protein database or in an S100-β–specific database. Unique m/z values were identified using Findpept (http://web.expasy.org/findpept/) to select those that could be derived from S100-β. Potential unique peptide epitopes of 8–10 aa long were chosen.

Chromatography was undertaken on an LC-20AD series HPLC system (Shimadzu, Kyoto, Japan) equipped with a vacuum degasser, a binary pump, an autosampler, and a thermostat column oven. Separation of Indo5 was carried out on a Biobasic-18 column (5 μm, 2.1 mm × 50 mm; Thermo Fisher Scientific) at a flow rate of 500 μl/min and column temperature of 30°C. The injection volume was 10 μl.