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Technological Innovation and Resources

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

Amelia C. Peterson, Jason D. Russell, Derek J. Bailey, Michael S. Westphall and Joshua J. Coon
Molecular & Cellular Proteomics November 1, 2012, First published on August 3, 2012, 11 (11) 1475-1488; https://doi.org/10.1074/mcp.O112.020131
Amelia C. Peterson
From the ‡Departments of Chemistry and Biomolecular Chemistry, and Genome Center of Wisconsin, University of Wisconsin – Madison, Madison, Wisconsin 53706
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Jason D. Russell
From the ‡Departments of Chemistry and Biomolecular Chemistry, and Genome Center of Wisconsin, University of Wisconsin – Madison, Madison, Wisconsin 53706
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Derek J. Bailey
From the ‡Departments of Chemistry and Biomolecular Chemistry, and Genome Center of Wisconsin, University of Wisconsin – Madison, Madison, Wisconsin 53706
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Michael S. Westphall
From the ‡Departments of Chemistry and Biomolecular Chemistry, and Genome Center of Wisconsin, University of Wisconsin – Madison, Madison, Wisconsin 53706
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Joshua J. Coon
From the ‡Departments of Chemistry and Biomolecular Chemistry, and Genome Center of Wisconsin, University of Wisconsin – Madison, Madison, Wisconsin 53706
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  • For correspondence: jcoon@chem.wisc.edu
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  • Fig. 1.
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    Fig. 1.

    Schematic representation of SRM (A) and PRM (B) as performed on QqQ and QqOrbi (or QqTOF) instrumentation, respectively. In SRM (A), each product ion transition (white circle) is serially monitored (from 1 to 5) one at a time in distinct scans. In PRM (B), all product ion transitions (1–5, and all possible product ions, shown as black circles) are analyzed/monitored in one concerted, high resolution and high mass accuracy mass analysis. Q1 and Q3 refer to the first and third mass-resolving quadrupoles of the QqOrbi (Q1 only) and QqQ, and q2 to the quadrupole (or cell, in the QqOrbi case) in which beam-type CAD is performed. The isolation widths employed for each device in both experimental and theoretical data are given below each respective device. C, Theoretical comparison of the rate of correctly identifying a target peptide (as true positive rate in percent, TPR) from all theoretically possible peptides in the human tryptic peptidome in SIM and reaction monitoring experiments in which 1, 2, and 3 y-ion transitions (labeled as 1T, 2T, and 3T, respectively) are monitored for Orbitrap or TOF instruments (±5 ppm) and QqQ (±250 ppm) for the 25 peptides used in this study in their light and heavy forms (50 total peptides). Count refers to the average number of possible confounding species, including the target peptide, represented by the boxplots. TPR is calculated as 100/count.

  • Fig. 2.
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    Fig. 2.

    Extracted PRM score chromatograms (XSC, ±5 ppm, gray) overlaid with XICs (±5 ppm, blue; 7-point boxcar smoothed) and single-scan PRM spectra for peptide AETLVQAr (#17) isolated at ±1 Th under neat (left) and matrix-containing (right) conditions from 2 pm to 200 nm. Red dotted line in XSC plots represents the score acceptance threshold (8) for this peptide. Product ions detected in each spectrum are highlighted and the spectral score is labeled. Yellow arrows in each XIC/XSC plot indicate the retention time at which the associated single-scan spectrum was acquired. Hashed area in XIC/XSC plots designates the retention time period during which peak elution was expected based on the ±3σ range around the average retention time observed in 200 nm analyses.

  • Fig. 3.
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    Fig. 3.

    A, Comparison of QqOrbi PRM detection at ±1 Th with QqQ SRM for peptide GVSAFSTWEk (#1) at 200 pm and 2 nm in the presence of matrix. Transition XICs are shown for the entire duration over which the peptide was targeted, with a zoom-in on the relevant time period in the QqQ SRM case (from the region shaded in gray in the chromatograms at the far right). Additional y-type product ions present in the PRM data are shown in gray. XICs were extracted at ±5 and ±250 ppm for PRM and SRM, respectively. The maximum spectral score attained at each concentration in PRM is also labeled. B, Lowest concentration detected (as number of attomoles of peptide on column) for each peptide in neat and matrix-containing experiments for the 14 peptides targeted in both SRM and PRM.

  • Fig. 4.
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    Fig. 4.

    Comparison of linearity as %RSD and adjusted %RSD (supplemental Equation S1) for the 14 shared peptides targeted in QqOrbi PRM experiments (y axis) and QqQ SRM (x axis) under neat and matrix-containing conditions. Solid horizontal lines and dotted vertical lines represent the mean %RSD value, also labeled in the plot, of the associated data set. Data falling in the gray region demonstrate greater linearity in PRM experiments. Data in the white region demonstrate greater linearity in SRM experiments.

Tables

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    Table I Peptides and instrument parameters used in this study

    c - carbamidomethylcysteine (+ C2H3NO), r - heavy labeled arginine (13C615N4), k - heavy labeled lysine (13C615N2).

    #PeptideaPRM parametersSRM parameters
    Mono. massParent m/zSched. RT (min)Parent m/zTransition m/zCE (%)Sched. RT (min)
    1GVSAFSTWEk1118.5488560.281728.0–33.0560.584658.6462635.0–89.0
    805.821
    963.977
    2HFLTLAPIk1046.6368524.325726.5–31.0
    3ARPAcVDAr1024.5112513.26530.0–10.0
    4SGWTcTQPGGr1281.5753608.773813.0–20.0
    5EGQLAAGTcEIVTLDr1741.8544871.934529.0–36.0872.4321174.2453037.5–89.0
    1245.323
    1316.402
    6LWSLAEIATSDLk1476.5364727.902740.0–53.0
    7SEDEDEDEGDATr1648.8118739.27557.0–14.0
    8DIQFGSQIk1042.5539522.284223.0–29.0522.557540.5552529.0–37.5
    687.731
    815.861
    9HTGTPLGDIPYGk1436.6875682.358422.5–27.0
    10NSWGTDWGEk1215.5330594.264025.5–30.5594.580743.7082729.0–37.5
    800.760
    986.971
    11FSDLTEEEFr1362.7023641.794927.0–31.5642.134719.6752829.0–89.0
    820.762
    933.920
    12SFEGTDYGk1010.4436506.229113.0–20.0506.491591.5562522.0–29.0
    648.608
    777.723
    13TLNGIQLAr994.5799498.297221.0–26.5498.544610.6632622.0–35.0
    667.715
    781.818
    14AFSQNSVLIk1113.6274557.821022.5–27.0
    15WPGYLNGGr1028.5067515.260626.5–31.0515.532689.6772529.0–89.0
    746.729
    843.845
    16GALDGEAPr894.4435448.229010.0–20.0448.441539.498231.0–29.0
    654.586
    767.745
    17AETLVQAr896.4955449.255010.0–20.0449.471483.478231.0–29.0
    596.636
    697.741
    18FLNPEWk940.4898471.252226.5–31.0
    19LEQNPEESQDIk1453.7908719.351010.0–20.0
    20SEASSSPPVVTSSSHSr1186.5135571.275610.0–15.0571.569583.511261.0–22.0
    771.693
    870.825
    21APcQAGDLr996.4686499.241610.0–15.0499.520541.514241.0–22.0
    669.644
    829.839
    22TWFQNQr988.4754495.245020.0–26.0
    23TVFSSTQLcVLNDr1710.8048825.413231.0–40.0825.891899.9732937.5–89.0
    1129.207
    1216.285
    24FSEVSADk889.4273445.720910.0–17.0445.944527.513221.0–29.0
    656.628
    746.706
    25GLYEGTGr861.4220431.718310.0–20.0
    • ↵a All peptides have a charge state (z) of +2, except for peptide #20 (z = +3).

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    Table II Comparison of QqQ SRM and QqOrbi PRM dynamic range and linearity
    SRMPRM
    ±0.2 Th±1 Th
    Means
        Neat
            # orders3.32.73.5
            %RSD42.0929.9927.36
            %RSDadj12.9711.807.72
        Matrix
            # orders2.22.42.9
            %RSD16.0929.9829.08
            %RSDadj8.0313.0710.49
    p values (SRM vs. PRM)
        Neat
            # orders2.61E-02b1.89E-01
            %RSD2.33E-02a8.74E-04a
            %RSDadj2.38E-011.99E-05a
        Matrix
            # orders5.47E-012.77E-03a
            %RSD2.72E-02b3.08E-02b
            %RSDadj1.05E-015.11E-01
    • ↵a PRM data show significantly greater linearity or dynamic range (α = 5E-02).

    • ↵b SRM data show significantly greater linearity or dynamic range (α = 5E-02).

Additional Files

  • Figures
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  • 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).
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Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics
Amelia C. Peterson, Jason D. Russell, Derek J. Bailey, Michael S. Westphall, Joshua J. Coon
Molecular & Cellular Proteomics November 1, 2012, First published on August 3, 2012, 11 (11) 1475-1488; DOI: 10.1074/mcp.O112.020131

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Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics
Amelia C. Peterson, Jason D. Russell, Derek J. Bailey, Michael S. Westphall, Joshua J. Coon
Molecular & Cellular Proteomics November 1, 2012, First published on August 3, 2012, 11 (11) 1475-1488; DOI: 10.1074/mcp.O112.020131
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Molecular & Cellular Proteomics: 11 (11)
Molecular & Cellular Proteomics
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