title: Functional microgels assisted tryptic digestion and quantification of cytochrome c through internal standard mass spectrometry.
abstract: Quantitation of cytochrome c (Cyt c) in cell lysates through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using gold nanoparticles (Au NPs) as the matrix and GR-10 peptide as an internal standard has been demonstrated. To shorten digestion time, temperature sensitive microgels containing trypsin (TR) and Au NPs have been employed. As-prepared functional microgels (TR/Au NPs/MGs) allow digestion of Cyt c within 15 s under microwave irradiation. The internal standard SALDI-MS approach provides linearity (R(2) = 0.98) of MS signal ratio (I 1168.6/I 1067.6) of the tryptic digested peptide (m/z 1168.6) to GR-10 peptide (m/z 1067.6) against the concentration of Cyt c ranging from 25 to 200 nM, with a limit of detection (at a signal-to-noise ratio of 3) of 10 nM. This approach has been validated by the analysis of the lysates of HeLa cells, with an average concentration of 13.7 ± 3.5 μM for cytoplasmic Cyt c. Increased concentrations of Cyt c in the HeLa cells treated with etoposide (a commercial drug) or carbon dots (potential drug) have been revealed through this simple, sensitive, and rapid SALDI-MS approach, supporting the drugs induced Cyt c-mediated apoptosis of the cells. This study has shown that this internal standard SALDI-MS approach holds great potential for cell study.
authors.affiliation: Department of Chemistry, National Taiwan University, Taipei, 10617, Taiwan.
authors.forename: Li-Yi
authors.initials: LY
authors.lastname: Chen
authors.forename: Wei-Cheng
authors.initials: WC
authors.lastname: Wu
authors.forename: Huan-Tsung
authors.initials: HT
authors.lastname: Chang
mesh_terms: D045304:Cytochromes c / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism; D006046:Gold; D006367:HeLa Cells; D006801:Humans; D053768:Metal Nanoparticles; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D006046:Gold; D045304:Cytochromes c; D014357:Trypsin