Separation Abilities of Capillary Electrophoresis Coupled with Ion Mobility Mass Spectrometry for the Discrete Detection of Sequence Isomeric Peptides
Round 1
Reviewer 1 Report
The manuscript Separations by Glazyrin et al. is reporting a study related to the separation of three peptides (isomers). The study is interesting considering the hyphenation of CE with MS.
The manuscript needs some revision before acceptance.
Remarks:
The state of the art should be implemented with additional references, e.g., published papers by Kasicka.
The English should be revised, e.g., in “Chemical”, the word purchased is reported several times in a short section.
Why authors did not report the separation of the three peptides using CE-MS? This electropherogram should be included.
Remarks about the use of sheath-liquid should be reported: the flow in this interface is very high compared to the CE flow. Most probably it will disturb the separation and the sensitivity.
Author Response
The state of the art should be implemented with additional references, e.g., published papers by Kasicka.
The additional references concerning the state of the art of the CE-MS and IMS-MS methods for separation and detection of isomeric peptides were added to the Introduction section.
The English should be revised, e.g., in “Chemical”, the word purchased is reported several times in a short section.
Major English corrections were made in the manuscript, as well as in the Chemicals section.
Why authors did not report the separation of the three peptides using CE-MS? This electropherogram should be included.
The CE-UV experiments have shown that the DR and RR peptides overlap on the corresponding electropherograms (Figures 1c, 1d), showing the inefficiency of their separation by CE. This result was also expected for the CE-MS experiments. To avoid overlapping of the DR and RR peaks, the peptides were separated by this method in pairs with the DD peptide (Figures 4, 5), and not in a mixture of three. Therefore, the CE-MS experiments with a mixture of all three peptides were not made according to the experimental design. In addition, the electropherograms in paired mixtures (Figures 4, 5) show the same release time of the DR and RR peptides, which additionally indicate their overlap in a possible experiment with all three peptides. Some comments about this point are included in the Results section. Unfortunately, the electropherograms with the three peptides cannot be presented without additional experiments hampered by the lack of sufficient amount of the synthetic peptides available for a limited time.
Remarks about the use of sheath-liquid should be reported: the flow in this interface is very high compared to the CE flow. Most probably it will disturb the separation and the sensitivity.
The sheath liquid in the CE-MS interface plays the role of an electrically conductive medium for creating an electrical potential during the CE. The sheath fluid rate in the CE-ESI interface was 1.2 μL/min, which greatly exceeds the estimated electroosmotic flow rate in the capillary (rated as about 73.6 nl/min). Indeed, in this case, the sensitivity can be reduced to a certain extent due to the dilution of the analytes. But in our case, the sensitivity turned out to be sufficient enough for the separate detection of the isomeric peptides, what was the achievement of the experimental goal. The difference between the peak capillary release times of the two peptides after separation was about 9 seconds (0.15 minutes). As for interference for separation, this time interval, in our opinion, is sufficient for their separate detection without interference from the sheath liquid. The necessary comments on these points have been added to the Results and Discussions section.
Reviewer 2 Report
The manuscript by Xu et al. entitled „Separation Abilities of Capillary Electrophoresis Coupled with Ion Mobility Mass Spectrometry for the Discrete Detection of Sequence Isomeric Peptides“ presents essential new data concerning applicability of capillary electrophoresis for peptide separation.
This is an interesting article for peptide biochemistry, but it is surprising why the authors chose the technically difficult approach instead of the standard sequenced cDNA. What are the advantages of direct peptide isomer analysis over sequencing of cDNA encoding this peptide?
Besides DNA sequencing there is also direct protein sequencing. The authors do not discuss this technique at all.
Author Response
It is surprising why the authors chose the technically difficult approach instead of the standard sequenced cDNA. What are the advantages of direct peptide isomer analysis over sequencing of cDNA encoding this peptide?
The presented techniques of capillary electrophoresis and ion-mobility mass spectrometry are widely used for protein and peptide separation and detection. But the most interest of the presented approach was to combine them into a single technical pipeline to test the abilities of separation and discrete detection of peptides with very similar masses. For this, the isomeric peptides specially synthesized artificially were used. Since their sequences were known in advance, the inverse problem of testing and proving the performance of the presented methods was solved for possible future works with unknown natural peptides in natural mixtures. To solve such a primary task, it is indeed possible to use coding DNA sequencing as an alternative. But in some cases the cDNA sequencing is not capable to trace changes in the peptide sequence due to alternative splicing occurring after the DNA transcription. Some notes about this point have been added to the Introduction section.
Besides DNA sequencing there is also direct protein sequencing. The authors do not discuss this technique at all.
Some remarks about the direct protein sequencing as an alternative to mass spectrometry were added to the Introduction section.
Round 2
Reviewer 1 Report
This reviewer has no further comments.
