Dhb matrix

The MALDIXin procedure was performed as previously described¹³ . Briefly, a single colony cultured on Mueller-Hinton agar (bioMérieux, La Balme-les-Grottes, France) was resuspended in 200 μl of distilled water, washed three times with double distilled water and resuspended in 100 μl of double distilled water. 0.4 μL of the bacterial solution was loaded onto the target and immediately overlaid with 0.8 μL of a 2, 5-dihydroxybenzoic acid (DHB) matrix (Sigma Aldrich, Gillingham, United-Kingdom) used at a final concentration of 10 mg/mL in chloroform/methanol (CHCl₃/MeOH) 90:10 v/v. Bacterial solution and matrix were mixed directly on the target by pipetting and the mix was dried gently under a stream of air (less than one minute). MALDI-TOF MS analysis was performed on a 4800 Proteomics Analyzer (Applied Biosystems, Foster City, USA) using the reflectron mode. Samples were analyzed by operating at 20 kV in the negative ion mode using an extraction delay time set at 20 ns. Mass spectrometry data were analyzed using Data Explorer version 4.9 (Applied Biosystems).

The 1.5 mL Eppendorf® Safe-Lock microcentrifuge tubes, potassium hydroxide (KOH), potassium borohydride (KBH₄), glacial acetic acid, methanol (MeOH), super DHB matrix (2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid; 9:1), mucin from bovine submaxillary glands (BSM) type I-S, iodomethane (ICH₃) and the Parafilm® M sealing film were obtained from Sigma (Dorset, UK). Human colorectal cancer cell lines SW480, SW620 and LS174T, and human pancreatic cancer cell lines PaTu S and PaTu T were obtained from the Department of Surgery at Leiden University Medical Center (Leiden, The Netherlands). The 96-well release plates (4ti-0125), the PCR plates, the foil pierce seals, the semi-automatic heat sealer (HT121TS), the polypropylene collection plates and the silicone plate lids were purchased from 4titude (Surrey, UK). HT permethylation kit (LT-PERMET-VP96) and the cation exchange cartridges (LC-CEX) were obtained from Ludger Ltd (Oxfordshire, UK). The ultrasonic bath (Bandelin Sonorex Digitec DT103H) was from Schalltec (Frankfurt, Germany). Samples were dried down in a Savant centrifugal evaporator from Thermo (Hampshire, UK). All automated steps in the analytical workflow described were performed using a Hamilton MICROLAB STARlet Liquid Handling Workstation from Hamilton Robotics Inc. (Bonaduz, Switzerland).