Quantitative analysis of newly synthesized proteins

Abstract

Measuring proteome response to perturbations is critical for understanding the underlying mechanisms involved. Traditional quantitative proteomic methods are limited by the large numbers of proteins in the proteome and the mass spectrometer’s dynamic range. A previous method uses the biorthogonal reagent azidohomoalanine (AHA), a methionine analog, for labeling, enrichment and detection of newly synthesized proteins (NSPs). Newly synthesized AHA proteins can be coupled to biotin via CuAAC-mediated click chemistry and enriched using avidin-based affinity purification. The combination of AHA-mediated NSP labeling with metabolic stable isotope labeling allows quantitation of low-abundant, newly secreted proteins by mass spectrometry (MS). However, the resulting multiplicity of labeling complicates NSP analysis. We developed a new NSP quantification strategy, called HILAQ (heavy isotope–labeled azidohomoalanine quantification), that uses a heavy isotope–labeled AHA molecule to enable NSP labeling, enrichment, identification and quantification. In addition, the AHA-peptide enrichment used in HILAQ improves both the identification and quantification of NSPs over AHA-protein enrichment. Here, we provide a description of the HILAQ method that includes procedures for (i) pulse-labeling and harvesting NSPs; (ii) addition of biotin by click reaction; (iii) protein precipitation; (iv) protein digestion; (v) enrichment of AHA-biotin peptides by NeutrAvidin beads and four-step elution; (vi) MS analysis; and (vii) data analysis for the identification and quantification of NSPs by ProLuCID and pQuant. We demonstrate our HILAQ approach by identifying NSPs from cell cultures, but we anticipate that it can be adapted for applications in animal models. The whole protocol takes ~6 d to complete.

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Fig. 1: Flowchart of the protocol, with procedure step numbers.
Fig. 2: Schematic diagram of the differences between the traditional quantitative proteomic strategy and HILAQ.
Fig. 3: HILAQ performed in an HT22 oxytosis model.
Fig. 4: Analysis of 108 cell death pathway–enriched proteins with twofold change after 1 h of labeling in HT22 cells as compared with HEK293T cells.

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Acknowledgements

We thank C. Liu from the Institute of Computing Technology, Chinese Academy of Sciences, for help with the use of pQuant. We thank C. Delahunty and X. Meng for critical reading of the manuscript. This work was supported by funding from the National Institutes of Health (P41 GM103533, R01 MH067880, R01 MH100175 to the Yates laboratory).

Author information

Affiliations

Authors

Contributions

Y.M., D.B.M. and J.R.Y. designed the research. Y.M. prepared samples, performed the MS analysis and analyzed the data. S.B. and W.W.W. contributed to hAHA synthesis. Y.M. wrote the manuscript.

Corresponding author

Correspondence to John R. Yates III.

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Competing interests

J.R.Y. declares that he is a consultant for Cambridge Isotope Laboratories. W.W.W. and S.B. declare that they work for Cambridge Isotope Laboratories, which sells the hAHA. The remaining authors declare no competing interests.

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Related link

Key reference using this protocol

1. Ma, Y. et al. J. Prot. Res. 16, 2213–2220 (2017) https://doi.org/10.1021/acs.jproteome.7b00005

Integrated supplementary information

Supplementary Figure 1 A typical MS1 spectrum of HILAQ samples.

Top: one MS1 spectrum with m/z range from 400 to 2000. Bottom: enlarged m/z range (560-660). The blue framed area indicated one pair of hAHA and AHA peptides (mass difference of 6)

Supplementary Figure 2 Box plot analysis on every replicate of HILAQ and QuaNCAT using Graphpad Prism (v5.01).

The box plot was created with Turkey whiskers. Adapted with permission from Ma et al.14, American Chemical Society

Supplementary Figure 3 Comparison of NSPs identified by HILAQ, QuaNCAT or QuanNCAT-pep.

Three independent biological replicates had been performed using each strategy. Error-bar represents “Mean with SD”

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–3, Supplementary Table 1 and Supplementary Manuals 1 and 2

Reporting Summary

Supplementary Table 2

Identification results for HILAQ and QuaNCAT

Supplementary Table 3

List of proteins with twofold change

Supplementary Data 1

pQuant parameter file: element

Supplementary Data 2

pQuant parameter file: aa

Supplementary Data 3

pQuant parameter file: modification

Supplementary Data 4

Completed pQuant parameter setting file

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Ma, Y., McClatchy, D.B., Barkallah, S. et al. Quantitative analysis of newly synthesized proteins. Nat Protoc 13, 1744–1762 (2018). https://doi.org/10.1038/s41596-018-0012-y

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