The submitted observation is mostly well written and has sufficient technical detail. Elucidating the molecular and functional characteristics of proteins is indeed tricky and time consuming and the authors successful pairing of 2D gel electrophoresis and LC-MS/MS has yielded a number of results; this was published in a previous paper which they do refer to. I understand it is a follow up, and the switch from 3-11NL to 3 – 5.6NL will of course increase the resolution and allow for clearer separation of proteins, especially those whose mass and isoelectric points are similar. The authors successfully resolve these proteins and are able to demonstrate their positions on 2D gel.
Technically, the methods are sound and indeed have been published before by the authors or others - it appears most of what they present here has been published before. I appreciate they are showing the proteins together on the same gel, however given that most of this is already published, I fear it detracts somewhat from the novelty and the impact.
A very minor point:
The authors cite the use of the malarial antigen PfTRAP as an argument for the use of these proteins as potential vaccine targets, and they state a reference to the clinical trial where PfTRAP was used in combination with RTS,S/AS02 in an attempt to boost the efficacy of RTS,S, however they do not talk about the result of the trial which were published in 2014. The combination of PfTRAP and RTS,S/AS02 failed to boost efficacy and indeed all volunteers challenged had circulating parasitemia. This highlighted the importance of adjuvant choice as the formulation used failed to elicit a strong CD8+ response. This is a common feature of the immunity, or lack thereof against many protozoa, as antigens are poorly immunogenic and/or require high titers to confer protection. Whilst I am aware this is not the focus of the manuscript, the addition of PfTRAP/RTS,S does seem like a quick justification for why these proteins are important, but they have not read the recent literature. This could be amended or removed should the manuscript be accepted.