Co-Immunoprecipitation-Coupled Mass Spectrometry Analysis of Zyxin’s Interactome and Phosphosites in Early Xenopus laevis Development

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this work, the authors analyzed zyxin protein interactions and phosphorylation dynamics during early development of Xenopus laevis embryos using Co-IP coupled with mass spectrometry. Authors identified stage-specific changes in zyxin-binding partners, including integrins, actin regulators, kinases, and transcription factors. The study revealed novel phosphorylation sites and confirmed zyxin’s role in cytoskeletal signaling pathways. This is an interesting finding and can be accepted for publication upon addressing the following questions:

Major concerns:

1. What is the functional relevance of phosphorylation changes in zyxin? Can authors validate phosphtylation sites using antibodies or mutations?
2. How do zyxin interactome dynamics in Xenopuscompare to mammalians. Are these evolutionarily conserved?
3. Include heat map or bar chart of zyxin interactome dynamics across stages
4. Western blot showing zyxin precipitation at different stages

 

 

Minor concerns

 

5. How were Xenopus laevis embryos staged and processed for Co-IP experiments?
6. How were phosphopeptides quantified across developmental stages?
7. Can you map Phosphorylation sites map on zyxin structure

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript used co-immunoprecipitation-coupled MS to analyze Zyxin in different stages of frog embryo development. I have the following concerns:

Major comments:

1. No MS raw data is provided. Without any supporting information, many conclusions in the current manuscript cannot be justified. Please provide the MSMS data for key peptide fragments in the SI so that protein identity or counts can be verified.
2. The current manuscript needs careful rereading. The format is very inconsistent, and there are many typos.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript provides a comprehensive analysis of zyxin-containing protein complexes and phosphorylation patterns during early Xenopus laevis embryogenesis. The combined use of Co-IP with both DDA- and DIA-based MS is methodologically solid, and the authors successfully identify developmental stage–specific interactome changes, particularly among integrins, cytoskeletal elements, kinases, and transcriptional regulators (Ybx, Gli1). The identification of differential phosphorylation at several conserved serine residues further strengthens the biological relevance of the study. The manuscript is generally well written and logically structured. However, several issues need to be addressed before publication.

Major concern:

1). Overinterpretation of mechanistic significance: The manuscript frequently interprets Co-IP/MS results as evidence of direct functional changes (e.g., signaling pathway regulation, mechanotransduction dynamics, apoptosis control). However, Co-IP from embryo lysates cannot distinguish: direct vs. indirect interactions; dynamic complex remodeling vs. biochemical stability; changes in protein abundance vs. binding affinity.

2). Lack of Validation Experiments: Although the authors present extensive DDA and DIA proteomic data, the manuscript lacks any orthogonal validation of the identified interactions or phosphorylation sites. All conclusions rely solely on mass spectrometry. At minimum, Co-IP/WB validation of key partners (integrins, Ybx1/2, Gli1, 14-3-3, AKT2) is essential. The functional implications of the phosphorylation patterns (Ser197/198, Ser383/386) also require validation using phospho-deficient mutants or phospho-specific antibodies. Without these experiments, the biological significance remains speculative and the conclusions are not sufficiently supported.

Miner concern:

1). Several grammatical errors and typos need correction (e.g., “Ingibitors”).

2). Methods should be in more details. What's the volume or protein amount of antibody used per IP reaction? What is the approximate total protein yield obtained from 100 embryos?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have answered all my questions . The paper is now ready to be accepted. 

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing my comments. I have no further concern.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed my concerns, and I recommend publication.