As 1 autosampler

Using an Eksigent AS-1 autosampler connected to a Tempo nanoLC system (Eksigent, Dublin, CA, USA) as previously described [13 (link)], 10 µL of each combined extract sample was loaded at 20 µL/min with MS buffer A (2% Acetonitrile + 0.2% Formic Acid) onto a C8 trap column (Michrom Bioresources, Auburn, CA, USA). After washing the trap for three min, the peptides were washed off the trap at 300 nL/min onto a PicoFrit column (75 µm ID × 150 mm; New Objective, Woburn, MA, USA) packed with Magic C18AQ resin (Michrom Bioresources, Auburn, CA, USA), then eluted from the column and into the source of a QSTAR Elite hybrid quadrupole-time-of-flight mass spectrometer (AB Sciex, Eksigent, Dublin, CA, USA) using the following program [14 (link),15 (link)]: 5%–30% MS buffer B (98% acetonitrile + 0.2% formic acid) over 60 min, 30%–80% MS buffer B over 3 min, 80% MS buffer B for 2 min, 80%–85% for 3 min. The eluting peptides were ionised at 2300 V. An Intelligent Data Acquisition (IDA) experiment was performed, with a mass range of 350–1500 Da continuously scanned for peptides of charge state 2+–5+ with an intensity of more than 30 counts/s. The selected peptides were fragmented and the product ion fragment masses measured over a mass range of 100–1500 Da. The mass of the precursor peptide was then excluded for 120 s.

These methodologies were performed as described previously [21 (link),22 (link)]. Briefly, samples were loaded using an Eksigent AS-1 autosampler connected to a Tempo nanoLC system (Eksigent, USA) at 20 µl min⁻¹ onto a C8 trap column (Michrom, USA) before washing and elution at 300 nl min⁻¹ onto a PicoFrit column (75 µm × 150 mm) packed with Magic C18AQ resin (Michrom, USA). Peptides were eluted and ionized into the source of a QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer (AB Sciex) at 2300 V using the following programmes: 5−50% MS solvent B (98% acetonitrile + 0.2% formic acid) over 30 min for gel slices or 15 min for gel spots, 50−80% MS B over 5 min, 80% MS B for 2 min, 80−5% for 3 min. An intelligent data acquisition experiment was performed, with a mass range of 350−1500 Da scanned for peptides of charge state 2+ to 5+ with an intensity of more than 30 counts scan⁻¹. Selected peptides were fragmented, and the product ion fragment masses were measured over a mass range of 50−1500 Da. The mass of the precursor peptide was then excluded for 120 s for gel slices or 15 s for gel spots.