Peptide Detection and Structure Determination in Live Cells Using Confocal Raman Microscopy

Peptides are an important class of bioactive compounds that continue to be developed for a variety of therapeutic uses. The bioactivity of peptides stems in most cases from their ability to enter or bind to the surface of cells to elicit a cellular response, and the primary sequence and secondary structure of the peptide determine this. Therefore, experimental methods that can provide structural information on peptides in live cells are useful for exploring peptide structure–activity relationships and metabolism directly within the targeted cellular environment. In this chapter we describe an experimental methodology for the detection and structure determination of exogenous peptides within living cells using confocal Raman microscopy (CRM). CRM is Raman spectroscopy performed under a confocal microscope. Raman spectroscopy itself has been applied to the study of peptides for several decades and provides a wealth of information, including secondary structure via the amide backbone vibrational modes, cysteine redox status via the S–S and S–H stretches, and disulfide conformation via the S–S stretch. The Raman spectra of peptides are dominated by intense bands associated with the aromatic ring vibrations of Phe, Tyr, and Trp. The positions and intensities of some of these bands are sensitive to the hydrophobicity and pH of the peptide environment and thus can potentially be used as intracellular probes. Heavy-isotope labeling of aromatic ring side chains shifts the spectral positions of the aromatic ring vibrations and enables unambiguous detection of the peptide within cells. We employ this method primarily for the study of cell penetrating peptides in live cells. However, the method could in principle be applied to the study of any type of peptide within any type of cell if the intracellular concentration of the peptide reaches high enough levels to enable detection.