Systematic evaluation of quantotypic peptides for targeted analysis of the human kinome

Abstract

In targeted proteomics it is critical that peptides are not only proteotypic but also accurately represent the level of the protein (quantotypic). Numerous approaches are used to identify proteotypic peptides, but quantotypic properties are rarely assessed. We show that measuring ratios of proteotypic peptides across biological samples can be used to empirically identify peptides with good quantotypic properties. We applied this technique to identify quantotypic peptides for 21% of the human kinome.

Figure 1. Development of a targeted proteomic assay for human protein kinases.

(a) Overview of the workflow combining enrichment and identification of nucleotide binding proteins with subsequent evaluation of proteotypic and quantotypic peptides. (b) Bar graph showing the unique and accumulated number of identified kinases across six cell lines. (c) Box plots displaying the number of evaluated peptides per protein including total number of in silico digested peptides evaluated by SRM, all peptides identified by SRM, assigned proteotypic peptides, and proteotypic peptides validated by synthetic counterparts. For each box, centre lines show the medians; box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles and outliers are represented by circles. (d) Coverage of the human kinome. Kinases with successfully identified proteotypic peptides are shown in red. Kinases only detected by discovery MS are shown in yellow. The use of the kinome tree is reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

Figure 2. Systematic evaluation of quantotypic peptide properties.

(a) Diagram representing the approach underlying quantotypic evaluation. Multiple proteotypic peptides from the same protein are quantified across several samples. The relative ratios of each of these peptides are calculated and statistically evaluated by multiple linear regression analysis. Peptides with highly correlated relative abundance have good quantotypic behaviour. (b,c) Box plots representing the proportion of quantotypic peptides determined across a panel of 6 (b) and 12 (c) cell lines included in the resampling analysis. Correlation analysis with resampling was conducted for differing numbers of total cell lines. The fitted red line is a prediction from a cubic smoothing spline estimating the number of quantotypic peptides identified if additional cell lines were included. Box plots are defined as in Fig. 1. (d) Dot plot showing the proportion of quantotypic peptides per protein analysed, across all 12 cell lines. Kinases where no quantotypic peptides were identified are marked in red.

Figure 3. Relative quantification of kinases directly from total cell lysate using quantotypic peptides.

The relative abundance of each kinase is displayed in a heatmap, depicting the 83 protein kinases across six cell lines. The colour key represents the relative abundance (by row z-score). Kinases and cell lines are labelled and dendrograms from hierarchical clustering are plotted.