In this manuscript titled “Kinase activities in pancreatic ductal adenocarcinoma with
prognostic and therapeutic avenues”, Vallés-Martí et al. perform mass spectroscopy (MS)-based proteomic/phospho-proteomic analyses of 42 pancreatic ductal adenocarcinoma (PDAC) tumors. Their main finding is the identification of three subtypes each based on proteomic (Prot1-3) and phospho-proteomic (Phos1-3) characteristics. The authors perform an in-depth characterization of the subtypes characterized. Cross-correlation with subtypes reported in previous publications is commendable; I think this is very valuable to readers interested in this topic.

While I have some reservations whether MS-based proteomic/phospho-proteomic analyses will perform better than transcriptomic/genomic analyses in guiding therapeutic options, the data and analyses presented by the authors are an important and valuable contribution to those interested in many aspects of PDAC biology and therapy. Some aspects of the manuscript could be improved, as I detail below:

Major comments:
1, Overall, I felt the results section could be better organized. There is a lot of interesting information being discussed in each of the five result sub-sections, which somewhat makes it difficult to follow. Perhaps the authors could consider further sub-dividing the current sub-sections so that each sub-section corresponds to the main finding(s) being discussed. I also felt that the results sub-section titles (headings) are rather generic; may I kindly suggest (though not necessarily insist) that the authors re-consider the sub-section titles so that they succinctly summarize the main finding(s) being discussed in the subsection.
2, The discussion section seemed a little too long, for example with some redundant explanation already thoroughly presented in the results. Please shorten if possible.

Minor comments:
1, Materials & Methods, “Clinical samples”: It would be helpful if the authors also refer the reader to supplementary table 1 here.
2, Materials & Methods, “Sample preparation for phosphotyrosine enrichement”: The authors mention that for “phosphotyrosine immunoprecipitation (pY-IP) 2.2-2.5 to 5 mg protein was used”. I’m not totally sure I understand this: do the authors wish to say 2.2-5 mg, or something else? A clarification would be appreciated.
3, Results, “Protein and phosphoprotein landscapes of PDAC tumor tissue”: The authors discuss pY level evaluated by western blotting and refer the reader to Fig S3g. This probably should be Fig S3h.
4, Results, “Proteome characterization of PDAC tumours”: The authors mention that “Prot2 tumours were particularly enriched for both iCAF and myCAF markers” and refer the reader to Fig S6c. I do not think the figure to which the reader is being referred is correct. Please check.
5, Results, “Proteome characterization of PDAC tumours”: The authors mention Prot1/3 having high tumour cellularity and refer the reader to Fig. 1a-d. I was not entirely convinced from the heatmap in Fig 1a that Prot1/3 was particularly associated with high cellularity. A clarification would help.
6, Results, “Proteome characterization of PDAC tumours”: The authors at the end mention immune-suppressive environment of Prot2, but I was not sure of their reasoning. Please clarify.
7, Results, “Mutation status and phosphoproteomic landscape”: The authors claim that mutation status being notably different between phosphoproteome subtype and mention G12D being observed in 7/15 Phos2 versus 5/19 Phos3 whereas G12V being observed in 4/15 Phos2 versus 9/19 Phos3. Rather than showing the proportion of each mutant per phosphoproteome subtype, I feel it would be better to calculate the proportion of each phosphoproteome subtype for each mutation.
8, The captions for Fig S6 and S7 seem swapped (or the figures are reversed).

Thank you for the revisions. I felt the authors have overall sufficiently addressed the issues I have raised in my previous review.

Regarding Major Comment #7 in my pervious review, I believe there are still some issues that remain unresolved. If the authors recalculated the proportion of each phosphoproteome subtype for each KRAS mutation type, should not the denominator be the same for each fraction shown for a particular mutation? For example, let the total numbers of KRAS G12D and G12V mutations were A and B, respectively. Furthermore, let the number of KRAS G12D mutations found in Phos2 and Phos3 be w and x, and KRAS G12V mutations found in Phos2 and Phos3 be y and z, respectively. Then, I believe the corresponding text should be:
“KRAS G12D mutations were mainly observed in Phos2 (w/A) compared to Phos3 (x/A), whereas G12V mutations were mainly found in Phos3 (z/B) compared to Phos2 (y/B).”

Also, if I may just point out a minor typographical error: in the material & methods section (“Sample preparation for phosphotyrosine enrichment”), the authors mention “20 μM cryostat sections”. This probably should read “20 μm” instead.

Thank you for the revisions. The concerns I raised in my review reports have all been sufficiently addressed, and I feel the paper is now much strengthened.