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. 2016 Jul 28;166(3):766-778.
doi: 10.1016/j.cell.2016.06.041. Epub 2016 Jul 21.

Human SRMAtlas: A Resource of Targeted Assays to Quantify the Complete Human Proteome

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Human SRMAtlas: A Resource of Targeted Assays to Quantify the Complete Human Proteome

Ulrike Kusebauch et al. Cell. .
Free PMC article


The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.


Figure 1
Figure 1. Human SRMAtlas development
The scheme outlines the workflow steps to generate SRM assays for every human protein. Peptides were selected for 20,277 proteins in the UniProtKB/Swiss-Prot database as well as for spliced isoforms, SNPs and modifications. Selection of PTPs was an iterative process by mining MS observed peptides in PeptideAtlas and the use of prediction tools. The PABST algorithm evaluated sequence constraints and ranked observed and predicted peptides, the highest scoring peptides for each protein were selected for SRM assay development. Peptides were individually synthesized and pooled in sets of 96. Peptides were analyzed on an Agilent 6530 Q-TOF with five CEs to acquire high-resolution MS/MS spectra to create spectral libraries and CE plots. SRM coordinates were extracted from the spectral library to acquire chromatographic traces on an Agilent 6460 QQQ. SRM assays were also developed on a Sciex QTrap 5500, upon the detection of a transition a full MS/MS spectrum was acquired to create a QTrap spectral library. SRM assay parameters including precursor and fragment ion type, charge state and rank order, elution time as well as chromatograms, MS/MS spectra and CE plots are provided in the human SRMAtlas resource. The human SRMAtlas is integrated with external knowledge bases providing comprehensive information on a protein of interest. See also Figure S1 and Table S2.
Figure 2
Figure 2. Human proteome coverage
The graph details the number of peptides per protein by empirically observed peptides in the human PeptideAtlas (build 2010-05, blue) and by PTPs selected for the human SRMAtlas (red). ‘5+’ specifies five or more peptides. ‘any’ shows the number of proteins for which at least one peptide is available. 9946 proteins (49.1% of the predicted human proteome) were described by MS observed peptides in PeptideAtlas 2010. SRMAtlas provides with 99.9% proteome coverage for 20,255 proteins by synthetic peptides. See also Table S1 and Table S3.
Figure 3
Figure 3. SRM assay success
(A) Number of developed SRM assays per instrument type in comparison to the number of synthesized peptides. The 6530 Q-TOF extracted coordinates served as input for the 6460 QQQ derived SRM assays with a success of 84.9%. 6530 Q-TOF and QTrap 5500 combined result in 158,015 targeted assays constituting 95.1% of the selected peptides. (B–G) Selected peptides (red) and their assay success rate (blue) in percent are displayed by (B) peptide length, (C) expected charge state, (D) hydrophobicity as SSRCalc value, (E) amino acid, (F) N-terminal amino acid and (G) C-terminal amino acid. See also Figure S2.
Figure 4
Figure 4. SRM assay coverage in the human SRMAtlas
Assay coverage by peptides per protein and instrument is displayed in green shades, selected peptides are shown in grey. 158,015 successfully developed assays represent 99.7% (20,225 proteins) of the human proteome (dark green). 95.4% of the human proteome is presented by at least three assays. 22 proteins are inaccessible. See also Figure S3 and Table S4.
Figure 5
Figure 5. Drug-induced inhibition of cholesterol synthesis
Systematic proteomic quantification of proteins from a reported gene module and enzymes in the cholesterol synthesis pathway upon drug treatment. (A) The heatmap shows the change in protein abundance following the treatment with lipoprotein deficient serum (LPDS) and atorvastatin compared to control conditions (untreated and 0.01% DMSO) of the same cell line. The signal represents the mean result from three independent biological experiments and three SRM analyses per sample. Proteins were hierarchically clustered according to the elicited response with the Ward2 algorithm and euclidean distance. Based on the clustering tree and the protein regulation we defined five different clusters of proteins showing similar response (I–V). Proteins marked with asterisks were included as “housekeeping” proteins for normalization. (B) Shown are the measured proteins from the pathway synthesizing cholesterol from acetyl-CoA. The enzymes are sorted by their position in the pathway and the proteins are color-coded according to the cluster in (A) they belong to (I–IV). The proteins or metabolites in italics have not been measured and are included for completeness. The inhibition of HMGCR by atorvastatin and the negative feedback of SREBP2 are depicted. (C) SRM chromatograms of peptide TQNLPNC[160]QLISR and LFSASEFEDPLVGEDTER from protein FDTF1 showing the difference in signal abundance between untreated cells and cells treated with LDPS + 5 μM atorvastatin as representative examples. The lower signal in untreated cells is magnified in the inset. See also Table S5.
Figure 6
Figure 6. Network of proteins associated with docetaxel perturbation of the cell cycle
SRM-based quantification of a protein network in three prostate cancer cell lines, DU145, LNCaP and PC-3, at four time points post-treatment with docetaxel and in untreated controls in comparison to mRNA abundance changes. The microarray-derived functional network was visualized with Ingenuity Pathway Analysis (IPA). The structure of the network is based on the IPA Core Analysis, STRING and Pathway Commons derived direct interactions and indirect relationships. Each heatmap visualizes the log2 abundance change of treated versus control cells for each time in each cell line at the transcript (mRNA) and protein (SRM) level. The signal represents the mean result from two technical replicates at the transcript level and three SRM analyses per sample. See also Figure S4 and Table S5.

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