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Dynamics of Non-Canonical Amino Acid-Labeled Intra- and Extracellular Proteins in the Developing Mouse

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Abstract

Introduction

Mapping protein synthesis and turnover during development will provide insight into functional tissue assembly; however, quantitative in vivo characterization has been hindered by a lack of tools. To address this gap, we previously demonstrated murine embryos can be labeled with the non-canonical amino acid azidohomoalanine (Aha), which enables the enrichment and identification of newly synthesized proteins. Using this technique, we now show how protein turnover varies as a function of both time and cellular compartment during murine development.

Methods

Pregnant C57BL/6 mice were injected with Aha or PBS (control) at different embryonic time points. Aha-labeled proteins from homogenized E12.5 and E15.5 embryos were conjugated with diazo biotin-alkyne, bound to NeutrAvidin beads, selectively released, then processed for either SDS-PAGE or LC–MS/MS. For turnover studies, embryos were harvested 0–48 h after Aha injection at E12.5, separated into different cellular fractions based on solubility, and analyzed via western blotting.

Results

We developed an enhanced method for isolating Aha-labeled proteins from embryos that minimizes background signal from unlabeled proteins and avidin contamination. Approximately 50% of all identified proteins were found only in Aha samples. Comparing proteins present in both Aha and PBS samples, 90% were > 2-fold enriched in Aha-treated embryos. Furthermore, this method could resolve differences in the Aha-labeled proteome between developmental time points. Newly synthesized Aha-labeled proteins were observed by 3 h and peak labeling was around 6 h. Notably, extracellular matrix and cytoskeletal turnover appeared lower than the cytosolic fraction.

Conclusions

The methods developed in this work enable the identification and quantification of protein synthesis and turnover in different tissue fractions during development. This will provide insight into functional tissue assembly and ultimately inform the design of regenerative therapies that seek to promote growth and repair.

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Abbreviations

ACN:

Acetonitrile

AG:

Aminoguanidine

Aha:

Azidohomoalanine

C:

Cytosolic

CS:

Cytoskeletal

CuAAC:

Copper(I)-catalyzed azide-alkyne cycloaddition

DBA:

Diazo biotin-alkyne

DTT:

Dithiothreitol

ECM:

Extracellular matrix

FA:

Formic acid

FDR:

False discovery rate

GO:

Gene ontology

HPLC:

High performance liquid chromatography

LC–MS/MS:

Liquid chromatography–tandem mass spectrometry

LFQ:

Label-free quantification

M:

Membrane

Met:

Methionine

MS:

Mass spectrometry

N:

Nuclear

Na2S2O4 :

Sodium dithionite

NaAsc:

Sodium ascorbate

ncAA:

Non-canonical amino acid

QE HF:

Q exactive HF hybrid quadrupole-orbitrap mass spectrometer

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SILAC:

Stable isotope labeling by amino acids in cell culture

TBS:

Tris-buffered saline

TFA:

Trifluoroacetic acid

THPTA:

Tris(3-hydroxypropyltriazolylmethyl)amine

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Acknowledgments

The authors would like to thank Victoria Hedrick and Uma Ayal at the Purdue Proteomics Core. This work was supported by the National Institutes of Health [R21 AR069248, R01 AR071359 and DP2 AT009833 to S.C.] The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH. This work was also supported by the National Science Foundation [CAREER 1752366 to TKU]. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

Conflict of interest

Authors Aya Saleh, Kathryn Jacobson, Tamara Kinzer-Ursem, and Sarah Calve declare that they have no conflicts of interest.

Ethical Standards

All experimental protocols involving animals were performed in compliance with established guidelines and all methods were approved by Purdue Animal Care and Use Committee (PACUC, protocols# 1209000723 and 1801001682). PACUC requires that all animal programs, procedures, and facilities at Purdue University to abide by the policies, recommendations, guidelines, and regulations of the USDA and the United States Public Health Service in accordance with the Animal Welfare Act and Purdue’s Animal Welfare Assurance. Additionally, no human subjects research was conducted in this study.

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  1. Weldon School of Biomedical Engineering, Purdue University, 206 South Martin Jischke Drive, West Lafayette, IN, 47907, USA

    Aya M. Saleh, Kathryn R. Jacobson, Tamara L. Kinzer-Ursem & Sarah Calve

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Sarah Calve received her B.S. in Materials Science and Engineering from Cornell University, then a M.S. in Molecular, Cellular and Developmental Biology and a Ph.D. in Macromolecular Science and Engineering from the University of Michigan. Her doctoral research focused on the design and mechanical characterization of self-assembling constructs for musculoskeletal repair, under the guidance of Prof. Ellen Arruda. As a postdoctoral fellow at Northwestern University’s Children’s Memorial Hospital, Sarah investigated the role of extracellular matrix remodeling during newt limb regeneration. Sarah joined the Weldon School of Biomedical Engineering at Purdue University as an assistant professor in 2012. Her research group, the Musculoskeletal Extracellular Matrix Laboratory, is actively developing tools to quantify how the composition, turnover, organization and mechanical properties of the musculoskeletal system change during scar-free tissue assembly. The goal is to use these tools to elucidate how different components integrate to form functional tissues during normal development and identify parameters that will guide the design of regenerative therapies. In 2017, she became the first Purdue professor to receive the NIH Director’s New Innovator Award. Additional recognitions include the BMES-CMBE Rising Star Junior Faculty Award (2018), inclusion in the National Academy of Engineering, Japan–America Frontiers of Engineering Symposium (2018) and being named the Leslie A. Geddes Assistant Professor of Biomedical Engineering at Purdue (2019).

This article is part of the CMBE 2019 Young Innovators special issue.

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Figure S1.

Enrichment of Aha-labeled proteins using biotin-alkyne. (a) Western blot analysis of E15.5 embryos harvested from dams injected with 0.1 mg/g Aha or PBS. Soluble lysates (lysate) were reacted with biotin-alkyne using CuAAC (clicked) and isolated from unlabeled proteins using NeutrAvidin beads (2 mg protein was combined with 100 µL beads). Removal of unreacted biotin-alkyne via desalting columns (desalted) did not substantially affect total protein concentration. These conditions isolated the majority of Aha-labeled proteins from the lysates as indicated by minimal signal in the unbound lane. Aha-labeled proteins were eluted using 2 or 4% SDS (lanes 5 and 6 for PBS, lanes 11 and 12 for Aha, respectively). (b) LC–MS/MS analysis of the eluted proteins revealed a high percentage of avidin contamination. Average raw intensity of n = 2 embryos. (c) Non-specific binding of unlabeled proteins due to CuAAC. Control E15.5 embryos from PBS-injected dams were reacted using CuAAC (click) with diazo biotin-alkyne (DBA) and incubated with NeutrAvidin beads. Substantially more proteins were eluted off the beads when the CuAAC and DBA were present in the reaction (lane 2). Supplementary material 1 (EPS 3251 kb)

Figure S2. (a)

Chemical species used for CuAAC. (1) Aminoguanidine (AG); (2) tris(3-hydroxypropyltriazolylmethyl)amine (THPTA); (3) Diazo biotin-alkyne (DAB). (b) The diazobenzene group of DAB is cleaved by Na2S2O4 under neutral pH conditions. Supplementary material 2 (EPS 1215 kb)

Figure S3.

Summary of Aha-enriched proteins found at E12.5 (left) and E15.5 (right). (a) Scatter plot data from Fig. 2 separated by time point. (b) Heat map comparisons of Pearson Correlation coefficients indicated a higher degree of correlation between samples within the Aha and PBS groups. (c) Distribution of raw intensity of proteins found only in Aha as a function of cellular compartment, average of n = 3 biological replicates. Note the absence of Avidin (see also Fig. S1b). Supplementary material 3 (EPS 1388 kb)

Figure S4.

Representative westerns for all 5 fractions analyzed for Aha-labeling study in Fig. 3. Supplementary material 4 (EPS 31123 kb)

Supplementary material 5 (XLSX 23 kb)

Supplementary material 6 (XLSX 9 kb)

Supplementary material 7 (XLSX 762 kb)

Supplementary material 8 (XLSX 882 kb)

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Saleh, A.M., Jacobson, K.R., Kinzer-Ursem, T.L. et al. Dynamics of Non-Canonical Amino Acid-Labeled Intra- and Extracellular Proteins in the Developing Mouse. Cel. Mol. Bioeng. 12, 495–509 (2019). https://doi.org/10.1007/s12195-019-00592-1

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