Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics

Supplemental Data

Files in this Data Supplement:

• Supplemental Methods - SUPPLEMENTAL METHODS 1.1 Liquid Chromatography 1.2 Data analysis SUPPLEMENTAL REFERENCES SUPPLEMENTAL EQUATIONS
• Supplemental Figure 2 - Distribution of the spectral score (equation 1) for all possible combinations of b- and y-ions from an 8 amino acid peptide by the fraction of the score described by each ion.
• Supplemental Figure 3 - Number of experiments which yielded quantitative data over 0 concentration orders-of-magnitude (200 nM only), 1 order-of-magnitude (20-200 nM), 2 (2-200 nM), 3 (0.2-200 nM), 4 (0.02-200 nM), and 5 orders-of-magnitude (0.02-200 nM).
• Supplemental Figure 4 - Linearity, as %RSD, plotted versus the number of orders-of-magnitude considered in the linearity calculation normalized by the lowest concentration order-of-magnitude accepted as a positive detection for all experiment types.
• Supplemental Figure 5 - (A) Extracted SIM ion chromatograms (XIC, ±5 ppm; 7-point boxcar smoothed) and single-scan (unless otherwise noted) spectra for peptide SFEGTDYGk (#12) under neat (left) and matrix-containing (right) conditions from 2 pM to 200 nM (corresponding to 2 amol to 200 fmol on column with 1 μL injected in every experiment). (B) Comparison of relative noise levels of high and low mass accuracy XICs for peptide SFEGTDYGk (#12) isolated at ±2 Th under matrix-containing conditions from 2 pM to 200 nM.
• Supplemental Figure 6 - Average number of peptide-spectrum matches for triplicate data-dependent (top 10) analysis of a tryptic yeast digest at isolation widths from ±0.2 to ±1 Th.
• Supplemental Results and Discussion - SUPPLEMENTAL RESULTS AND DISCUSSION 2.1. PRM parameter evaluation. 2.2. Detection criteria for PRM and SRM. 2.3. Empirical comparison of QqQ SRM and QqOrbi PRM. 2.4. QqOrbi SIM detection criteria. 2.5. QqOrbi SIM measurement precision, dynamic range, and linearity. 2.6. Empirical comparison of QqOrbi SIM and QqQ SRM.
• Supplemental Tables 1 - Supplemental Table 1A. Extracted score chromatogram maxima for all neat PRM ±1 Th experiments. Supplemental Table 1B. Extracted score chromatogram maxima for all matrix PRM ±1 Th experiments. Supplemental Table 1C. Extracted score chromatogram maxima for all neat PRM ±0.2 Th experiments. Supplemental Table 1D. Extracted score chromatogram maxima for all matrix PRM ±0.2 Th experiments.
• Supplemental Tables 2 - Supplemental Table 2A. QqOrbi measurement precision by batch category. Supplemental Table 2B. QqOrbi measurement precision by concentration. Supplemental Table 2C. QqOrbi measurement precision by peptide.
• Supplemental Tables 3 - Supplemental Table 3A. QqOrbi dynamic range by batch category. Supplemental Table 3B. QqOrbi depression of dynamic range by experimental characteristics.
• Supplemental Tables 4 - Supplemental Table 4A. QqOrbi measurement accuracy by batch category. Supplemental Table 4B. QqOrbi measurement accuracy by individual experiment. Supplemental Table 4C. QqOrbi measurement accuracy by batch category, excluding lowest detected concentration. Supplemental Table 4D. QqOrbi measurement accuracy by individual experiment, excluding lowest detected concentration. Supplemental Table 4E. QqOrbi measurement accuracy by batch category, statistical comparison of all data versus data excluding lowest detected concentration. Supplemental Table 4F. QqOrbi measurement accuracy by individual experiment, statistical comparison of all data versus data excluding lowest detected concentration. Supplemental Table 4G. QqOrbi measurement accuracy, as adjusted %RSD, by batch category. Supplemental Table 4H. QqOrbi measurement accuracy, as adjusted %RSD, by individual experiment.
• Supplemental Tables 5 - Supplemental Table 5A. QqQ measurement precision by batch category and individual experiment. Supplemental Table 5B. QqQ measurement precision by concentration. Supplemental Table 5C. QqQ measurement accuracy and dynamic range by batch category and individual experiment.
• Supplemental Tables 6 - Supplemental Table 6A. Comparison of QqOrbi PRM and QqQ measurement precision data by batch category and individual experiment. Supplemental Table 6B. Comparison of QqOrbi PRM and QqQ measurement precision data by peptide and concentration-matched category for individual experiments. Supplemental Table 6C. Comparison of QqQ SRM and QqOrbi SIM dynamic range and linearity.
• Supplemental Figure 1 - Boxplots showing retention time reproducibility across all experiments and concentrations for each peptide targeted in QqQ and QqOrbi experiments (both SIM and PRM) under neat (blue) and matrix-containing conditions (red).