Noninvasive Imaging of Protein Metabolic Labeling in Single Human Cells Using Stable Isotopes and Raman Microscopy

• Published in: Analytical chemistry (Washington) Vol. 80; no. 24; pp. 9576 - 9582
• Main Authors: van Manen, Henk-Jan, Lenferink, Aufried, Otto, Cees
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.12.2008
• Subjects: Amino acids; Analytical chemistry; Cell culture; Chemistry; Deuterium - chemistry; Exact sciences and technology; HeLa Cells; Humans; Isotopes; Methionine - metabolism; Microscopy; Phenylalanine - metabolism; Proteins; Proteins - metabolism; Spectrometric and optical methods; Spectrum Analysis, Raman; Tyrosine - metabolism; Deuterium; Human; Tyrosine; Cell culture; Isotope labelling; Phenylalanine; Methionine; Protein; Tissue; Spatial distribution; Aminoacid; Imaging; Raman spectrometry; Intracellular
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac801841y

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.