Next-Generation Dried Blood Spot Samplers for Protein Analysis: Describing Trypsin-Modified Smart Sampling Paper
Round 1
Reviewer 1 Report
The different papers were tested based on DMS-paper unit and applied for bottom-up protein (digestion-involving) sample preparation workflow.
The methodology of DMS involved simple use of the different paper type on which the trypsin was captured serving to digest protein to peptides, subjected to mass spectrometry analysis.
The outcome would provide the effective binding of trypsin what was confirmed by analysis of Cytochrome C protein model compound
REMARKS
Does sampling of blood on paper refer to all DBS? There are materials except for papers, serving for effective spotting of biological samples?
What do you mean that DBS are minimally invasive? I understand the “non-invasive method” to be the one which contain no penetration of the living body, i.e., the procedure does not need any penetration step to e.g., blood circulation at all.
You do not mention/cite one important work dealing with progress in DBS regarding automation of DBS of small compounds. It is an indisputable part of the state-of-the art of DBS in its current development. Please, follow the text upgrade
TEXT UPGRADE
At present there are numerous publications and applications on the use of DBS for small biologically active compounds. Currently, the analysis of small biologically active compounds from DBS has become a routine tool which can be automated and combined with high-end analytical measurements [5, https://doi.org/10.1016/j.jchromb.2018.06.037].
Please, draw the analytical workflow. It would benefit the paper to make a quick glance for the reader seeing nicely and easily drawn scheme of the procedure.
In conclusion, please add some future plans in this scope of study, regarding its applicability to the routine clinical practice and how do you see to involve the suggested approach among the non-invasive methods. Would that be possible to innovate to make this approach in the way to measure on the spot?
Author Response
- Reviewer 1
”Does sampling of blood on paper refer to all DBS? There are materials except for papers, serving for effective spotting of biological samples?”
Response: to avoid the issue of defining the materials which can be used for DBS, we have rephrased the introducing sentence (page 3, ln 46-47):
”Sampling blood on paper for the determination of biologically interesting compounds has a history of more than a century. The use of dried blood spots (DBS) has the advantage that there is no need for trained medical….”
”What do you mean that DBS are minimally invasive? I understand the “non-invasive method” to be the one which contain no penetration of the living body, i.e., the procedure does not need any penetration step to e.g., blood circulation at all.”
Response: we have rephrased this sentence in the manuscript and avoid the confusing term ”minimally invasive” (page 3, ln 47-49):
“The use of dried blood spots (DBS) has the advantage that there is no need for trained medical personnel for sampling as it only requires a fingerprick for blood collection. It can be performed at home, even…”
”You do not mention/cite one important work dealing with progress in DBS regarding automation of DBS of small compounds. It is an indisputable part of the state-of-the art of DBS in its current development. Please, follow the text upgrade
TEXT UPGRADE
At present there are numerous publications and applications on the use of DBS for small biologically active compounds. Currently, the analysis of small biologically active compounds from DBS has become a routine tool which can be automated and combined with high-end analytical measurements [5, https://doi.org/10.1016/j.jchromb.2018.06.037].”
Response: the mentioned reference fits good in the introduction and is included (page 3, ln 60-61, reference 6)
”Please, draw the analytical workflow. It would benefit the paper to make a quick glance for the reader seeing nicely and easily drawn scheme of the procedure.”
Response: an analytical workflow is included as figure 7 as well as a legend to this figure (pg 24, ln 607).
”In conclusion, please add some future plans in this scope of study, regarding its applicability to the routine clinical practice and how do you see to involve the suggested approach among the non-invasive methods. Would that be possible to innovate to make this approach in the way to measure on the spot?”
Response: an additional part is added to the text under the heading 3.4. Future perspectives (page 25, ln 632 – 649).
“3.4. Future perspectives
Although the experiments and results described are purely fundamental in nature, we believe smart samplers have potential in routine clinical analysis. The smart samplers integrate time-consuming sample preparation in the sampling and transport and by such they will reduce the overall sample processing time and improve the sample through-put. In the specific case of trypsination very complex samples are generated from blood and serum on the modified paper and high-end analytical instrumentation for peptide separation and determination still will be needed. One potential use in clinical practice might be in personal proteomic profiling where larger populations sample and send their blood samples for general proteomic analysis. It also can be used in routine clinical practice for a targeted approach using LC-SRM-MS/MS when arrays of high- and medium abundant proteins are analysed simultaneously. For the lower abundant proteins it can be combined with peptide immunoaffinity clean-up and enrichment followed by LC-SRM-MS/MS determination. From an exploratory point of view, these smart samplers can function as small reactors which can be integrated in a proteomic workflow.
In a broader perspective, coupling of reagent proteins (other enzymes, antibodies) might even allow not only for sample preparation during sampling, but also for easy on-spot determination of certain target compounds.”
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comment: Minor Revision
This manuscript is well written and interesting. I think this should be published after minor revision.
Possibly authors would consider to clarify the following issues given below:
1) Introduction is structured in an unconventional manner and it would be fine if authors can restructured it.
2) I am not quite happy with the quality of some of the figures used in the manuscript and few of them should be redraw and plotted again in a high resolution and reader friendly manner.
Figures should be changed given below:
Figure 2: Redraw as a high resolution figure using a software such as ChemDraw or any other suitable software. Current figure looks like it is copied from somewhere else.
Figure 4: X and Y axis should be bold and clear to reader (including A and B figure).
Figure 5: Labels on X and Y axis are not visible properly and hard to read. Labels should be easy to read and clear. I would suggest please make them larger and bold so that readers can understand them in a proper manner.
Figure 6 : Figure legend is not written to the bottom of the figure.
Author Response
RESPONSE TO THE REVIEWERS
- Reviewer 2
”Possibly authors would consider to clarify the following issues given below:
1) Introduction is structured in an unconventional manner and it would be fine if authors can restructured it.”
Response: the introduction is restructured by moving some sentences and deleting others. Check the ”marked version” of the revised manuscript to review these changes. Page 3 and 4, ln 46 – 122.
”2) I am not quite happy with the quality of some of the figures used in the manuscript and few of them should be redraw and plotted again in a high resolution and reader friendly manner.
Figures should be changed given below:
Figure 2: Redraw as a high resolution figure using a software such as ChemDraw or any other suitable software. Current figure looks like it is copied from somewhere else.
Figure 4: X and Y axis should be bold and clear to reader (including A and B figure).
Figure 5: Labels on X and Y axis are not visible properly and hard to read. Labels should be easy to read and clear. I would suggest please make them larger and bold so that readers can understand them in a proper manner.”
Response: all figures have been upgraded to better resolution. Both in the manuscript and in the supplementary. information. Figure 4 (page 18): the labels are made more clear. Figure 5 (page 21): the labels are made more clear.
”Figure 6 : Figure legend is not written to the bottom of the figure.”
Response: the legend has been placed at the bottom of the figure (page 23)
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
I consider to publish manuscript at current form.
