Computational protocol: Index-ion Triggered MS2 Ion Quantification: A Novel Proteomics Approach for Reproducible Detection and Quantification of Targeted Proteins in Complex Mixtures*

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Protocol publication

[…] RAW files from the LTQ-Orbitrap were converted to mzXML by ReAdW (version 4.3.1) with default parameters except whenever ITA was used the “precursorFromFilterLine” option was applied.For the ITA experiment, the acquired MS2 spectra were searched against a yeast database (yeast.nci.20080206; 11,319 entries searched) supplemented with the sequences of the targeted peptides using X!Tandem 2 (2007.07.01.3). The following search parameters were applied: full tryptic cleavage specificity; mass tolerance of ± 20 ppm for precursor ions and ±0.4 Da for fragment ions; no missed cleavage allowed; fixed modification on Cys (+45.9877 Da) and variable modifications on Met (+15.9949), Lys (+148.1092), and N termini (+140.095). Peptide identification was achieved by processing the search results with the Trans-Proteomic Pipeline (TPP, http://tools.proteomecenter.org/wiki/index.php?title = Software:TPP).For the MSTIQ assay, the seven datasets were each searched twice against the mouse International Protein Index database (v.3.56;56,294 entries searched) using X!Tandem2 as described above with the following parameter changes: a fixed modification of +57.021464 on Cys was used; one search was conducted with fixed modifications on N termini (+144.1021) and Lys (+144.1021) to account for HL peptide modifications; the other search was conducted with fixed modifications on N termini (+140.095), Lys (+148.1092), and Arg (+3.98814) to account for LH peptide modifications. Each search was repeated against the reversed database to estimate the false positive rate. Peptide identification was achieved by processing the search results with the TPP. Confidently identified peptides (fully-tryptic, 2+ and 3+ ions with no missed cleavages, PeptideProphet probability ≥ 0.9) in at least one of the seven titers were considered target peptides.Each target peptide was quantified in all seven datasets using ISBquant (available on request). ISBquant considered a scan for quantification of a target peptide if 1) the theoretical and observed parent m/z's were within 20 ppm, and 2) at least 7 HL or 7 LH fragment ions in the MS2 scan were observed above background level (BG, defined as the 25th percentile of all non-zero intensities in the scan) within 0.5 units of their predicted m/z's. This enabled some peptides to be quantified in titers in which they did not pass the PeptideProphet threshold of 0.9. Scan selection was further restricted by identifying the highest quality scan, and only scans with a retention time ≤ ±1 min from the highest quality scan were used for quantification. For this purpose, scan quality was judged by the number of observed HL or LH MSTIQ ions (whichever was greater); ties were broken by the median observed MSTIQ ion intensity in each scan. In addition to automatic scan selection (applied in ), ISBquant allows for manual scan selection by the user.To compute peptide abundance ratios, the intensities of each fragment ion pair i were quantified as xi=ln(intensity(HLi)intensity(LHi)) and outliers were detected with the “MAD-Median Rule” (see reference (), p.101): xi is an outlier if |xi − M| > 3.321*m; the choice of the constant 3.321 corresponds to a 5% chance of rejecting a nonoutlier from a normally distributed sample), where M = median{xi} and m = MAD(xi) = 1.4826*median{|xi − M|} denotes a robust estimate of the standard deviation based on the sample's Median Absolute Deviation (R mad() function). The final quantification for each identified peptide is mean {xi} following rejection of outliers. This “trimmed” mean estimate has known standard error s=sw.95n, where sW is the sample Winsorized variance (see reference (), p.63). Finally, peptides were only considered “quantified” if at least two MSTIQ-labeled fragment ion pairs contributed to the trimmed mean. A MSTIQ ion was considered “observed” if its intensity ≥ SNR*BG; unless otherwise noted, SNR = 2. Some potential MSTIQ fragment pairs were excluded from quantification for one of four reasons: (1) all b1+ and y1+ fragment ion pairs were excluded, (2) any fragment ion pair containing a MSTIQ ion with a predicted m/z within ±1.5 Da of another predicted MSTIQ ion, or a neutral loss from a predicted ion (b-ions, loss of NH3; y-ions, loss of H2O), was excluded, (3) any fragment ion pair for which the intensity of at least one MSTIQ ion was not “observed” was excluded. A fragment ion pair was deemed quantifiable if the intensity of one ion (HL- or LH-derived) was above background and the intensity of the sibling ion was nonzero.For the iMSTIQ assay automated scan selection for quantification is achieved using the same constraints described for MSTIQ. In addition, the index ion elution profiles were used to assist in scan selection. Ion intensities for the predicted monoisotopic index ion peak (±20 ppm), as well as intensities for peaks at +1/z and −1/z were extracted. The peak at −1/z is because of the slight impurity of the heavy isotopes used in labeling (see Discussion). Elution of an index ion was distinguished from other ions based on two criteria. First, a scan was considered to contain an index ion if intensities of 0.05–0.35× (-1/z peak) and 0.5–1.5× (+1/z peak) the predicted index ion's monoisotopic peak intensity were present. Second, only MS2 scans triggered from MS1 scans with an index ion intensity of ≥10% of the peak index ion elution intensity were considered. MS2 scans without this evidence of an intense, co-eluting index ion were rejected. Other than these differences, scan selection and quantification were the same as for MSTIQ. Scans were selected automatically for the iMSTIQ study in and entirely manually for the macrophage study in . […]

Pipeline specifications

Software tools ReAdW, TPP, PeptideProphet
Databases IPI
Application MS-based untargeted proteomics