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Plug-and-play analysis of the human phosphoproteome by targeted high-resolution mass spectrometry

Nature Methods volume 13, pages 431–434 (2016)Cite this article

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Abstract

Systematic approaches to studying cellular signaling require phosphoproteomic techniques that reproducibly measure the same phosphopeptides across multiple replicates, conditions, and time points. Here we present a method to mine information from large-scale, heterogeneous phosphoproteomics data sets to rapidly generate robust targeted mass spectrometry (MS) assays. We demonstrate the performance of our method by interrogating the IGF-1/AKT signaling pathway, showing that even rarely observed phosphorylation events can be consistently detected and precisely quantified.

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Figure 1: A database for targeted human phosphoproteome analysis.
Figure 2: Plug-and-play assay performance.

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Acknowledgements

We would like to thank S.A. Gerber for providing data, the MacCoss lab for advice designing and analyzing DIA and PRM assays with Skyline, and the Villén lab for critical discussions. This work was supported by a Samuel and Althea Stroum Endowed Graduate Fellowship to R.T.L., an Interdisciplinary Training in Genome Sciences grant from NIH/NHGRI (T32 HG00035) to B.C.S., a Howard Temin Pathway to Independence Award from NIH/NCI (K99/R00CA140789) to J.V., and an Ellison Medical Foundation New Scholar Award (AG-NS-0953-12) to J.V.

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Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Robert T Lawrence, Brian C Searle, Ariadna Llovet & Judit Villén

Authors
  1. Robert T Lawrence
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  2. Brian C Searle
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  3. Ariadna Llovet
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  4. Judit Villén
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Contributions

R.T.L. and J.V. conceived the study. R.T.L., B.C.S., and J.V. designed the experiments. R.T.L. and B.C.S. performed the experiments and analyzed data. A.L. created the web resource. J.V. supervised the work. R.T.L., B.C.S., and J.V. wrote the paper.

Corresponding author

Correspondence to Judit Villén.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Database coverage comparison.

(a) Number of phosphosites detected, PSM-level FDR 1%, before correcting for data aggregation. (b) Effect of data aggregation on phosphosite FDR. Since PSM-level target-decoy data were not accessible from PhosphoSitePlus, we estimated site-level “local FDR” from our data using the number of phosphosite PSM’s as the scoring metric. Sites only observed one time (singletons) are likely to be observed by random chance in aggregated data (> 40% in this case). (c) Database composition compared to PhosphoSitePlus. To control FDR, we compared the overlap of sites observed at least 5 times in each database.

Supplementary Figure 2 Analysis of most frequently observed phosphopeptide forms.

(a) Distribution of preferred cleavage states with sequential LysC/Trypsin digest or Trypsin only. (b) Enrichment versus background of amino acids in the miscleavage+1 position of preferred peptides. (c) Mass-to-charge distributions of predicted peptide charge states grouped by lower than observed, equal to observed or greater than observed. (d) Inter-laboratory cleavage form consensus. Phosphoisoforms observed at least once by 4 different laboratories were analyzed (n=7,897). The most frequently observed peptide representing a unique phosphoisoform for each study was considered the preferred sequence. The preferred sequence for each of the 4 studies was compared. For example “4-0” indicates that the preferred sequence was the same for all 4 studies.

Supplementary Figure 3 Targeted comparison of two cleavage states of ACLY phosphorylation at S455.

The isoform with two miscleavages is observed with greater than 10-fold higher intensity in than the fully cleaved isoform.

Supplementary Figure 4 Benchmarking PRM versus DIA and DDA.

(a) Correlation of observed retention times to database retention times (normalized to a 0-100 scale in Skyline). (b) Comparison of sampling efficiency of DDA, DIA, and PRM using either spectrum-centric (database searching) or peptide-centric (targeted signal extraction) approaches. DDA was not analyzed using the peptide-centric approach (e.g. by MS filtering).

Supplementary Figure 5 Analysis of phosphosite positional isomers.

(a) The number of observed versus theoretical positions by bin. In peptides with multiple phosphorylatable residues, typically more than half of the possible sites have been observed. Only singly-modified underlying phospho-peptide sequences observed at least 100x were analyzed (n=12,357). (b) Extracted fragment ion chromatogram of two singly phosphorylated isobaric peptides (FLMECRNS*PVTK and FLMECRNSPVT*K) resolvable by retention time.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 2385 kb)

Supplementary Table 1

Retention time prediction (XLSX 698 kb)

Supplementary Table 2

Peptide selection benchmarking (XLSX 29 kb)

Supplementary Table 3

Comparison of 101 phosphopeptides analyzed by DDA, DIA, and PRM (XLSX 1814 kb)

Supplementary Table 4

Label-free PRM analysis of IGF-1/AKT signaling (XLSX 28 kb)

Supplementary Data

Phosphopeptide sequence and normalized retention time database (ZIP 60565 kb)

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Lawrence, R., Searle, B., Llovet, A. et al. Plug-and-play analysis of the human phosphoproteome by targeted high-resolution mass spectrometry. Nat Methods 13, 431–434 (2016). https://doi.org/10.1038/nmeth.3811

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