Peer review of: Single molecule secondary structure determination of proteins through infrared absorption (2020)

Reviewers' comments:
Reviewer #1 (Remarks to the Author):
The manuscript by Ruggeri and co-authors reports on structural determination of single proteins
using AFM-IR. Specifically, the aurthors showed that using this highly sensitive technique they
could probe secondary structure of thyroglobulin and apoferritin determining amounts of a-helix,
b-turns and coil in their structures. The manuscript is well-written and should eventually be
accepted to the publication. However, prior to it, the authors must address the most important
question of their work that is not entirely clear. How do the researchers know that they dealt with
one protein molecule rather than with a protein aggregate. The reviewer suggests to use
alternative analytical technique, such as DLS, to convince the audience that single molecules of the
mentioned above proteins were studied. Once demonstrated, the reviewer suggests to accept the
manuscript.
Reviewer #2 (Remarks to the Author):
The article by Ruggeri et al addresses the investigation of isolated large biomolecules by the
photothermal AFM-IR technique. For increased sensitivity a slightly off-resonant measurement
modus was used in a study of isolated apoferritin and thyroglobulin molecules on a gold substrate.
Conclusions on the secondary structure of the investigated proteins are drawn with the
interpretation of measured AFM-IR and supplementary CD spectra.
Although the thyroglobulin molecules are with dimensions of about 8 nm x 10 nm x 5 nm quite
large, to my knowledge this is the first single molecule study by an mid-infrared spectroscopic
technique. As such new experimental IR spectroscopic possibilities give access to the analysis of
structural and chemical properties of single large biomolecules and macromolecules, they could be
in particular relevant for biomedical research. I recommend the publication of the article after
reasonable revision.
Major points:
I) How do you know that really only one molecule was measured? Please comment on the tip
convolution effects in the text.
II) It is claimed that physical properties of thermomechanical detection and field enhancement are
unraveled, where?
III) Potential future possibilities of the technique should be discussed better. In this context: e.g.,
could the internal structure of the large biomolecules be studied? Why is it not studied yet? It is
written that AFM-IR has spatial resolutions from about 1 to 10 nm. This should be maybe sufficient
to investigate e.g. the two parts of the homodimer thyroglobulin?
IV) What was the spatial resolution in the presented experiments? Please add. If the spatial
resolution was below 10 nm, why can the molecules not be studied without probing any parts of
the bare gold substrate? Please comment.
V) With respect to the used measurement modus, why didn’t the authors use the shown higher
order resonance at around 400 kHz in Fig. 1b? For this one protein and gold resonance should be
better separated than for the used resonance frequency at 194 kHz.
VI) Make a statement if the spectrum of the substrate is influenced by the QCl emission profile
related background as shown in Fig S1. How does this background and low sensitivity in particular
influence the amide 2 band shape in the spectra of the proteins, can this be related to the
significant differences between FTIR and AFM-IR spectra as seen in Figure 4. If not, what is the
reason?
 Minor:
 - Introduction: Thus, the IR absorption ... are not affected by plasmonic effects.
 I guess plasmonic resonances are meant here?. Please clarify.
 - Result sections:
 Add the technical details of the used mesh filter.
 - Caption Figure 1: ... high values can cause instabilities ..
 Comment: .. Maybe the signal is saturated?..
 - Figure 2 b: as it can be barely identified or mistaken please mark the measurement line by an
 other colour, e.g. green.
 - Materials section:
 What means a 20 μM of apoferritin.. ; solution?
 - Please control referencing of Ref. [32] after (Figure 4b) in the Results section.
 - A reference for the assignments used in the deconvolution of the amide I band (supplementary
 part) is missing.
 Supplementary:
 - 1st sentence: typo: ... we aimed at we aimed ....
 - Line 33: 10 mW is given as the lower limit for typical power of the laser. This sounds quite high
 for the study of an organic samples.
 - Line 56: contact resonance at higher angle?
 - A reference for the assignments used in the deconvolution of the amide I band (supplementary
 part) is missing.
 Reviewer #3 (Remarks to the Author):
Here are my comments regarding the manuscript:
 Manuscript untitled: « Single Molecule Secondary Structure Determination of Protein through Off-
 Resonance and Short Pulse Infrared Absorption Nanospectroscopy »
 I don’t recommend its publication in nature communication: it’s not innovative enough and the
 format is not appropriated to this technical article.
 Here are some reasons and the list is not comprehensive.
  General comments:
 The authors introduce off-resonance, low power and short pulse AFM-IR to acquire infrared
 absorption spectra of thyroglobulin at the single molecule level, with a high signal-to-noise ratio.
 Without any doubt, it’s essential to the community to have access to new protocols to improve the
 sensitivity and reliability of this technique but it’s not innovative enough to be published in a
 journal like nature communication.
 I don't want to minimize the interest of this publication. Since the analysis of single protein using
 AFM-IR is a real challenge, I suggest to the authors to publish it in a more specialized journal
 where they can bring a real discussion, develop the different ambiguous points and illustrate it
 with more figures. This way, I think it should be appreciated to its full value by giving more details
 (by merging the main text and the supplementary documents) and complementary information
 and by highlighting the results with more proteins.
 -You said “To date, however, the sensitivity of AFM-IR has been limited to the measurements of
 large (>0.3 m) and flat (~2-10 nm) self-assembled monolayers or biomolecular aggregates
 composed of hundreds of molecules and has not demonstrated the ability to characterise single
 molecules”: check other publications and update your bibliography.
 Some points are a little bit confusing:
 -Figure 1 d) is really difficult to understand. Are you sure of your legend (blue dots 100ns ? red
 dots 7%) I don’t understand the link.
 -Figure e): if your system is well optimized (in term of power and pulse width) the amplitude of
 the resonance of the substrate should be lower than the one of the protein. It’s not the case. Why?
 -in the caption of the figure 1 “(e) IR amplitude as a function of frequency pulse for signal on gold
 and on a thyroglobulin molecule at a laser power of 7% and at 1655 cm-1” the power used for the
 experiments is 7 % but in the text it’s written p5 that “already at the low laser power of 0.5 mW
 (1-5% of commercial IR sources), the enhancement of the field causes non-linearity and
 instabilities” may be I have missed something but the power used for the experiments should be
 lower than 7% ? I suppose it’s 7% of the power already reduce after the addition of the filter. I
 think it’s better to use only mW and not %.
 -Then figure 2 and 3 no power nor pulse width is indicated.
 -What about the discrepancies observed between FTIR results and AFM-IR? For example why is
 your amide II lower? May be there is a physical reason behind it: discuss it.
 But my main concern is about the interpretation of the local IR spectra: “Apoferritin has an -
 helical content of 75% (Figure 4a),34 while thyroglobulin has major contributions from -helix and
 coils (55%), -turn (17%) and intramolecular -sheet structures (28%) (Figure 4b).” How you can
 propose such repartition without showing a curve fitting? the reference extract from FTIR curve
 fitting (reference) should be in the main document.