Probot microfraction collector

This was done on a Thermo Scientific Dionex UltiMate^™ 3000 RSLC nano system (Sunnyvale, CA, USA). The analytical column was a capillary Acclaim PepMap RSLC (Dionex, Sunnyvale, CA, USA) C18 column (300 μm × 15 cm, 2 μm) that was kept at 30 °C and operated at a flow rate of 4 μL/min. The guard/trap column was a C18 PepMap 100 μ-precolumn (300 μm × 5 mm, 5 μm). Mobile phase A was water with 0.05% TFA and mobile phase B was acetonitrile. The trap column mobile phase was 1% acetonitrile with 20 mM TEAA. The samples were maintained at 5 °C in the autosampler. The Urine Extract (5 μL) was injected and the mobile phase was as follows: 4 min with 20 mM TEAA / 1% acetonitrile at 20 μL/min on the trap column (sent to waste), followed by a linear gradient after switching from 10% to 80% solvent B in 100 min, held at 80% of B for 4 min, returned to 10% B in 1 min, and re-equilibrated at 10% B for 3 min. UV absorbance detection was monitored at 220 nm, 260 nm and 320 nm. Collection of the eluate as 20 sec droplets was done with a Probot Microfraction Collector (Dionex) onto a blank MALDI plate (Opti-TOF 384 Well Insert, 123 × 81 mm, AB SCIEX, Framingham, MA, USA). CCA matrix (0.5 μL of 5 mg/mL in 50% acetonitrile with 7 mM ammonium phosphate dibasic to suppress salt adducts) was added to each spot manually using an Eppendorf Research Pro electronic pipette.