Selective TiO2 Phosphopeptide Enrichment of Complex Samples in the Nanogram Range
, Fanni Bugyi 1,3,†, Simon Sugár 1,4, Goran Mitulović 5,6, Károly Vékey 1, Lilla Turiák 1 and László Drahos 1,*Round 1
Reviewer 1 Report
This work describes an analytical proteomics research work, which aimed at optimization of a protocol for phosphopeptide enrichment from digests of protein complex samples in a sub-microgram concentration range. The experimental work was based on the use of spin tips containing titanium dioxide or a monolitic column with TiO2 nanoparticles. The authors focused namely on selectivity and performance of the two stationary phases when testing multiple loading, washing and elution solvent systems and displacing agents to eliminate non-specific binding of non-phosphorylated peptides. Selectivity comparisons took into consideration the following aspects: degree and site of phosphorylation, peptide length, isoelectric point and hydrophobicity. The best overall results were obtained with the spin tips and citric acid as displacing agent. In general, the enriched samples showed much better repeatability as regards to the number of identified phosphopeptides.
The manuscript is well written and its content is adequate to describe all experiments, which have been done. I only miss a paragraph in the discussion/conclusions which would put the present experiments and the resulting performance and selectivity parameters into the context of other research studies on phosphopeptide enrichment.
Minor points:
I suggest not to use the term “buffer” for solutions, which have been used for conditioning, loading, washing and elution. As these solution contained strong acids and large proportion of an organic component, they do not fulfil the definition of a buffer. I suggest to use the term “solvent” throughout the manuscript.
Page 2, lines 45-47, if this text part really refers to chromatographic fractionation of proteolytic peptides in general and not specifically to phosphopeptides, then it should mention reversed-phase peptide separation at the first position.
Page 11, line 306, there is a reference to text section 2.2.2, which does not exist.
Author Response
Dear Reviewer,
thank you for your positive opinion, comments, and corrections. Please see the attachment for the answers.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
I have carefully examined the manuscript entitled “Selective TiO2 phosphopeptide enrichment of complex samples in the nanogram range” by Tóth et al. In this paper the authors describe the development of a procedure for phosphopeptide enrichment, based on TiO2-based affinity chromatography, for application in complex samples containing low quantities of peptide mixtures.
Authors give an extensive and convincing description of the method, investigating several parameters, such as the enrichment factor, recovery, repeatability, and so on, needed for optimization of the performance phosphopeptide enrichment in complex samples. They also compared the different performance of the spin tips filled with TiO2 microspheres and a TiO2 nanoparticle coated monolithic columns, along with the use of several loading buffers. On this basis, TiO2 spin tips and the use of citric acid as a loading buffer gave the best results in terms of phosphopeptide recovery from samples in the low nanogram range.
I have much appreciated the design of the method, the experimental work and data analysis. Moreover, manuscript is written in a good English style, and don’t need language revisions.
The work could be considered a useful contribution in the context of the analysis of phosphorylated proteins. On this basis, I consider the manuscript suitable for publication on Separations in the present form.
Author Response
We thank the reviewer for the effort on evaluating our manuscript. We are grateful for his/her positive opinion.
