| Popis: |
State-of-the art liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis uses a constant electrospray (ESI) voltage for data acquisition. Modern qualitative and quantitative LC-MS/MS methods depend on highly efficient gradient elution chromatography. The changing chemical composition of mobile phase during gradient elution results in an inherent disconnect with single point ESI voltage optimization. A constant ESI voltage limits spray stability and compromises chromatographic peak area quantification, limiting total peak area and increasing peak area relative standard deviation (RSD). We have adapted a previously reported closed-loop “orthogonal” feedback control system for qualitative proteomics† to one suitable for targeted peptide quantification. A digitally controlled nanospray source was modified to use the high-voltage output from an external, computer controlled power supply. A software program controlling the nanospray emitter stage position and the external power supply supported a programmable spray compensation voltage during chromatograpghic analysis. One microliter of a standard peptide (MRFA) was autosampler injected (CTC PAL) directly on column (New Objective PicoFrit, 75 um × 10 cm, C18) to the source equipped linear ion trap mass spectrometer (Thermo Scientific, LTQ). Repetitive oncolumn injections at different (fixed) target ESI voltage settings yields a apparent compound dependant response curve in which total chromatographic peak area for selected ion currents reaches a maximum value. Spray stability and chromatographic quality was directly correlated with the observed spray mode. Altering the ESI voltage from 2 kV to 3 kV resulted in a peak area increase of 38%. Qualitative visual comparison of noise in the molecular ion signal (523.5 m/z) showed a markedly higher level at 2.0kV. Fourier transform spectrum analysis of selected ion current, revealed a ten-fold increase in noise at 2 and 4 Hz for the 2 kV acquisition. Spray instability results in increased noise of the reconstructed chromatogram, increasing the uncertainly of peak area measurement. †Valaskovic, G.A, Murphy, J.P., Lee, M.S. “Automated Orthogonal Control System for Electrospray Ionization”, J. Am. Soc. Mass Spectrom., 2004, 15, 1201–1215. |
