Method and apparatus for analyzing biomolecules using Raman spectroscopy

A method for identifying the binding site of a protein bound to a low-molecular-weight compound, comprising the following steps of:

• (i) preparing fractionated fragments comprising the steps of:
• mixing the low-molecular-weight compound with the protein and obtaining the protein bound to the low-molecular-weight compound,
• fragmenting the protein bound to the low-molecular-weight compound by chemical degradation or an enzyme selected from the group consisting of protease and peptidase, and
• fractionating the fragmented protein bound to a low-molecular weight compound by liquid chromatography or capillary electrophoresis,
• (ii) subjecting fractionated fragments of the protein bound to the low-molecular-weight compound to Raman spectroscopy,
• (iii) detecting a fraction having a Raman peak derived from the low-molecular-weight compound bound to a fragment of the protein via Raman spectroscopy, wherein the low-molecular-weight compound comprises at least one type of substituent selected from the group consisting of an alkynyl group, a nitrile group, a diazonio group, an isocyanate ester group, an isonitrile group, a ketene group, a carbodiimide group, a thiocyanate ester group, an azide group, an alkynediyl group, and deuterium having a scattering spectrum in a silent region of the Raman spectrum and exhibits a Raman peak distinguishable from that of the protein, wherein said low-molecular-weight compound comprises said at least one type of substituent during said detection via Raman spectroscopy,
• (iv) subjecting the fraction subjected to Raman spectroscopy to mass spectrometry and tandem mass spectrometry, wherein said low-molecular-weight compound comprises said at least one type of substituent during said mass spectrometry and tandem mass spectrometry,
• (v) obtaining the mass and tandem mass spectrometric results for a fraction having a Raman peak derived from the low-molecular-weight compound,
• (vi) comparing the results with the mass information of the protein,
• (vii) identifying the fractionated fragment binding to the low-molecular-weight compound by searching for a fractionated fragment, whose mass is shifted by the low-molecular-weight compound, and
• (viii) identifying the amino acid residue within the protein which binds to the low-molecular-weight compound by searching for an amino acid residue, whose mass is shifted by the low-molecular-weight compound, thereby identifying the binding site of a protein, said method further comprising the steps of:
• adding a solution containing a metal nanoparticle or a metal nanostructure to the fractionated fraction prior to the Raman spectroscopy, and adding an organic acid to the fractionated fraction prior to the Raman spectroscopy.