Noninvasive imaging of protein metabolic labeling in single human cells using stable isotopes and Raman microscopy.
We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C-D stretching vibrational bands in these amino acids are observed in the 2100-2300 cm(-1) spectral region that is devoid of vibrational contributions from other, nondeuterated intracellular constituents. We found that incubation with deuterated amino acids for 8 h in cell culture already led to clearly detectable isotope-related signals in Raman spectra of HeLa cells. As expected, the level of isotope incorporation into proteins increased with incubation time, reaching 55% for deuterated phenylalanine after 28 h. Raman spectral imaging of HeLa cells incubated with deuterium-labeled amino acids showed similar spatial distributions for both isotope-labeled and unlabeled proteins, as evidenced by Raman ratio imaging. The SILAC-Raman methodology presented here combines the strengths of stable isotopic labeling of cells with the nondestructive and quantitative nature of Raman chemical imaging and is likely to become a powerful tool in both cell biology applications and research on tissues or whole organisms.
van Manen
Henk-Jan
HJ
Biophysical Engineering Group, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.
Lenferink
Aufried
A
Otto
Cees
C
D003903:Deuterium / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008715:Methionine / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000378:metabolism*
D011506:Proteins; D014443:Tyrosine; D010649:Phenylalanine; D008715:Methionine; D003903:Deuterium