Technical consistency (i.e. reproducibility and linearity) of the 3AA method was compared to two conventional chromatographic separations used for small molecule and amino acid analyses. Pancreatic cancer cell extracts (biological triplicates) either undiluted or diluted five-fold, were analyzed using (i) the 3AA method, as well as (ii) a 15 min gradient on a Kinetex HILIC column (150 × 2.1 mm i.d., 1.7 μm particle size – Phenomenex, Torrance, CA, USA) and (iii) a 23 min gradient on an Acquity UHPLC BEH Amide Column (2.1×100 mm, 1.7μm – Waters, Milford, MA, USA). In (ii), samples were analyzed on the Kinetex HILIC at 350 μl/min (mobile phases: A: ACN; B: 18 mΩ H2O, 20 mM (NH4)2CO3, 0.1% NH4OH; gradient: 1.5 min hold at 5% B; 5-60% B in 8.5 min; 60-95% B in 0.5 min at 0.5 ml/min; 95% hold for 2 min; 95-5% B in 0.5 min; 5 hold for 2 min; column temperature: 25°C). In (iii), samples were analyzed on the Acquity UHPLC BEH Amide Column (2.1×100 mm, 1.7μm – Waters, Milford, MA, USA) at 500 μl/min (mobile phases: A: 18 mΩ H2O, 20 mM (NH4)2CO3 pH 4.00; B: acetonitrile; gradient: 3 min hold 85% B; 85-30% B in 9 min; 30-2% B in 3 min; 2-85% of B in 1 min; 85% equilibration for 7 min; column temperature: 60°C).
The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in positive ion mode. Calibration was performed before each analysis using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, USA). Limits of detection (LOD) were characterized by determining the smallest injected amino acid amount required to provide a signal to noise (S/N) ratio greater than three using < 5 ppm error on the accurate intact mass. Based on a conservative definition for Limit of Quantitation (LOQ), these values were calculated to be three fold higher than determined LODs.
MS data acquired from the QExactive was converted from .raw file format to.mzXML format using MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed using a process for chemical formula determination using isotopic patterns and accurate intact mass (Clasquin et al. 2012 ). Analyte retention times were confirmed by comparison with external standard retention times, as indicated above.
Relative quantitation was performed by exporting integrated peak areas values into Excel (Microsoft, Redmond, CA, USA) for statistical analysis including T-Test and ANOVA (significance threshold for p-values < 0.05) and unsupervised Principal Component Analysis (PCA) (Pan et al. 2007 (link); Fonville et al. 2010 ), calculated through the MultiBase macro (freely available atwww.NumericalDynamics.com ).
The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in positive ion mode. Calibration was performed before each analysis using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, USA). Limits of detection (LOD) were characterized by determining the smallest injected amino acid amount required to provide a signal to noise (S/N) ratio greater than three using < 5 ppm error on the accurate intact mass. Based on a conservative definition for Limit of Quantitation (LOQ), these values were calculated to be three fold higher than determined LODs.
MS data acquired from the QExactive was converted from .raw file format to.mzXML format using MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed using a process for chemical formula determination using isotopic patterns and accurate intact mass (Clasquin et al. 2012 ). Analyte retention times were confirmed by comparison with external standard retention times, as indicated above.
Relative quantitation was performed by exporting integrated peak areas values into Excel (Microsoft, Redmond, CA, USA) for statistical analysis including T-Test and ANOVA (significance threshold for p-values < 0.05) and unsupervised Principal Component Analysis (PCA) (Pan et al. 2007 (link); Fonville et al. 2010 ), calculated through the MultiBase macro (freely available at
