Nanolc 415 system

An aliquot (5 μL) of each sample was loaded onto a Nano cHiPLC 200 μm ID × 0.5 mm ChromXP C18-CL 3 μm 120 Å reverse-phase trap cartridge (Eksigent, Dublin,CA) at 2 μL/min using an Eksigent autosampler. The bound peptides were flushed onto a Nano cHiPLC column 200 μm ID ×15 cm ChromXP C18-CL 3 μm 120 Å column (Eksigent) with a 15 min linear (5–95%) acetonitrile gradient in 0.1% formic acid at 1000 nL/min using an Eksigent 415 NanoLC system. The column was washed with 95% acetonitrile-0.1% formic acid for 5 min and then re-equilibrated with 5% acetonitrile-0.1% formic acid for 5 min. A 5600 Triple-Tof mass spectrometer (Sciex, Toronto, Canada) was used to analyze the metabolite profile. The IonSpray voltage for positive and negative modes were +/−2300 V and the declustering potential was +/−80 V. Ionspray and curtain gases were set at 10 psi and 25 psi, respectively. The interface heater temperature was 120 °C. Eluted compounds were subjected to a time-of-flight MS survey scan from m/z 50–1000 to determine the top twenty most intense ions for MSMS analysis. Product ion time-of-flight scans at 50 msec using a collision energy spread of 15 eV with a set collision point of 35 eV were carried out to obtain the tandem mass spectra of the selected parent ions over the range from m/z 50–1000. Spectra were centroided and de-isotoped by Analyst software, version 1.6 TF (Sciex).

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.