Im interested in the identification of the amide II band and its deuterated form for a proteomics experiment, however amide II is not usually visible in classical Raman spectrum, can i identify the band clearly with the SERS variant and a 532 nm laser?. Amide II can be easily detected with IR Spectroscopy, however, the concentration of the deuterated protein in my sample is very low and IR could not detect any signal. This problem can be overcome with SERS however i do not possess an UV Raman Spectrometer to make a resonance Raman experiment in wich the amide II band is clearly visible. If not possible, is there a different reliable parameter (Raman shift) in SERS to distinguish deuterated variants of a protein from other ones that are not labeled with deuterium?, i mean, perhaps an aminoacid sidechain or another amide band easily detected with an infrared, red or green laser?
Hi Oscar,
I do not know much about amide-II bands but a quick literature survey shows that it is indeed difficult to get a signal under non-resonant conditions. I have a few remarks:
1) Deuterating the protein should shift the characteristic bands to lower wavenumbers. For e.g. refer : Article In-situ observation of reactive wettability alteration using...
wherein we deuterate a surfactant (stearic acid) to differentiate its signal from the C-H bands present in mineral oil. Deuterating the stearic acid shifts its band (C-D2) to 2100 - 2300 cm-1, instead of 2800-3000 cm-1.2) I do not how low a concentration you want to detect, but efficient denoising techniques can help extract signals with low SNR. For e.g. refer : Article Algorithm-improved high speed and non-invasive confocal Rama...
in which we use principal component analysis based denoising to extract out signals which were invisible in the raw data. The paper also contains the matlab code used for denoising and maybe this can be useful for you.3) Finally, SERS often requires complex substrate fabrication and the obtained signals are very dynamic. I would recommend using graphene-enhanced Raman spectroscopy (GERS) for protein detection. The enhancement factors are modest (~ 103),, however, it is a relatively easier SERS technique. GERS has been used to detect 10 nM of analyte molecules from solution and has also been used for protein detection in blood. Please refer : Article Raman Enhancement of Blood Constituent Proteins Using Graphene
4) Finally, the GERS enhancement also depends on how well the analyte interacts with the graphene substrate. In your case, if the protein can have a side chain that is aromatic, this would lead to better sensitivity in Raman measurements because of pi-pi stacking between the aromatic side chain and the graphene lattice. The non-aromatic side chains will not give a clear signal in GERS. This could be a useful marker.
These are a few suggestions I can give to detect bands with low scattering cross-section. Good luck!
Dear Oscar Cienfuegos , as Sachin Nair I also don´t know much about amide -II bands, but if it is easily detactable by IR, you could give a try to ATR, where a really tiny amount of sample is required and the signal could be comparable or better than a conventional FTIR.
About SERS, and Raman in general, you should check that the mode you are looking for is also active in Raman /SERS. If the mode is active it should be possible to distinguish between deuterated and normal protein, because the vibration frequency should change.
Often in SERS you don´t need to deal with resonance, (SERRS), of course it is better, but conventional SERS can enhance enough the signal.
Hope it helps.
Please look at the following below links which may help you in your analysis:
https://clirspec.org/wp-content/uploads/2015/01/Wood.pdf
https://www.jove.com/pdf/52712/jove-protocol-52712-surface-enhanced-raman-spectroscopy-detection-biomolecules-using-ebl
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3586543/pdf/nihms-437835.pdf
Thanks
Hi Oscar Cienfuegos I think if you want a quick and less complex measurement of the amide II band you should opt for FTIR spectroscopy with an ATR attachment.
For my protein work, I used a PerkinElmer Spectrum Two that is a fairly popular FTIR tool. With the ATR attachment, you can simply drop 3-5 ul of protein solution onto the crystal (wiped clean first) and wait for the solution to dry (10-20 mins). Then you can take your measurements (order of seconds per spectrum usually). Although, if I am correct as you will have your protein in D20 you can make a liquid measurement since there will be little background contribution from H20. Analysis of the spectra can then be carried out using deconvolution/band fitting processes. There are several Researchgate discussion and publications that can help you with this.
I've just seen Manuel Gómez recommended the same thing! Hope this post helps to add to that. Best of luck with your protein study.
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