Quantitative top-down proteomics of SILAC labeled human embryonic stem cells.
Human embryonic stem cells (hESCs) are self-renewing pluripotent cells with relevance to treatment of numerous medical conditions. However, a global understanding of the role of the hESC proteome in maintaining pluripotency or triggering differentiation is still largely lacking. The emergence of top-down proteomics has facilitated the identification and characterization of intact protein forms that are not readily apparent in bottom-up studies. Combined with metabolic labeling techniques such as stable isotope labeling by amino acids in cell culture (SILAC), quantitative comparison of intact protein expression under differing experimental conditions is possible. Herein, quantitative top-down proteomics of hESCs is demonstrated using the SILAC method and nano-flow reverse phase chromatography directly coupled to a linear-ion-trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FT-ICR-MS). In this study, which to the best of our knowledge represents the first top-down analysis of hESCs, we have confidently identified 11 proteins by accurate intact mass, MS/MS, and amino acid counting facilitated by SILAC labeling. Although quantification is challenging due to the incorporation of multiple labeled amino acids (i.e., lysine and arginine) and arginine to proline conversion, we are able to quantitatively account for these phenomena using a mathematical model.
Collier
Timothy S
TS
W. M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
Sarkar
Prasenjit
P
Rao
Balaji
B
Muddiman
David C
DC
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
D002247:Carbon Isotopes; D020543:Proteome; D001120:Arginine; D008239:Lysine