415 autosampler

Using an Eksigent 415 autosampler connected to a 415 nanoLC system (Eksigent, USA), 5 µL (1 μg/μL) of the sample was loaded at 300 nl/min with MS buffer A (2% acetonitrile, 0.2% formic Acid) by direct injection onto a PicoFrit column (75 µmID × 150 mm; New Objective, Woburn, MA) packed with C18AQ resin, 1.9 µm 200 Å [Dr Maisch, Germany]. Peptides were eluted from the column and into the source of a 5600 TripleTOF hybrid quadrupole-time-of-flight mass spectrometer [Sciex, USA] using the following program: 2–35% MS buffer B (80% acetonitrile, 0.2% formic acid) over 90 min, 35–95% MS buffer B over 9 min, 95% MS buffer B for 9 min, 80–2% for 2 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+ to 5+ with an intensity of more than 400 counts/s. Up to 50 candidate peptide ions per cycle were selected and fragmented and the product ion fragment masses measured over a mass range of 100–2000 Da. The mass of the precursor peptide was then excluded for 15 s.

Using an Eksigent 415 autosampler connected to a 415 nanoLC system (Eksigent, USA), 5 µL of the sample was loaded at 300 nl/min with MS buffer A (2% Acetonitrile, 0.2% Formic Acid) by direct injection onto a PicoFrit column (75 µmID × 150 mm; New Objective, Woburn, MA) packed with C18AQ resin (1.9 µm, 200 Å; Dr Maisch, Germany). Peptides were eluted from the column and into the source of a 6600 TripleTOF hybrid quadrupole-time-of-flight mass spectrometer (Sciex, CA) using the following program: 2–35% MS buffer B (80% Acetonitrile, 0.2% Formic Acid) over 90 minutes, 35–95% MS buffer B over 9 minutes, 95% MS buffer B for 9 minutes, 80–2% for 2 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 400 counts/s. Up to 50 candidate peptide ions per cycle were selected and fragmented and the product ion fragment masses measured over a mass range of 100–2000 Da. The mass of the precursor peptide was then excluded for 15 seconds.

Using an Eksigent 415 autosampler connected to a 415 nanoLC system (Eksigent, Dublin, CA, USA), 1 µg /5 µL of the samples were loaded at 300 nL/min with MS buffer A (2% acetonitrile + 0.2% formic acid) by direct injection onto a self-made column with an integrated emitter made with a laser puller [63 (link)] (75 µm ID × 150 mm) and packed with C18AQ resin (1.9 µm, 200A, Dr. Maisch, Ammerbuch, Entringen, Germany). Samples were injected in biological and technical triplicates. Peptides were eluted from the column and into the source of a 6600 TripleTOF hybrid quadrupole-time-of-flight mass spectrometer (Sciex, Redwood City, CA, USA) using the following program: 2–35% MS buffer B (80% acetonitrile + 0.2% formic acid) over 90 min, 35–95% MS buffer B over 9 min, 95% MS buffer B for 9 min and 80–2% reverse concentration gradient for 2 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 400 counts/s. Up to 50 candidate peptide ions per cycle were selected and fragmented and the product ion fragment masses measured over a mass range of 100–2000 Da. The mass of the precursor peptide was then excluded for 15 s.