Accurate quantitation of glutathione in cell lysates through surface-assisted laser desorption/ionization mass spectrometry using gold nanoparticles Cheng-Kang Chiang, BS, Yang-Wei Lin, PhD, Wen-Tsen Chen, MS, Huan-Tsung Chang, PhD ⁎ Department of Chemistry, National Taiwan University, Taipei, Taiwan Received 8 October 2009; accepted 19 January 2010 Abstract We developed a method for the determination of three aminothiols—cysteine, glutathione (GSH), and homocysteine—using surface- assisted laser desorption/ionization mass spectrometry (SALDI-MS). The analytes were first captured using the unmodified 14-nm gold nanoparticles; N-2-mercaptopropionylglycine–modified gold nanoparticles serving as internal standard were sequentially added, and then the sample was analyzed using SALDI-MS. This approach provided good quantitative linearity of the three analytes (R 2 = ∼0.99), with good reproducibility (relative standard deviations: b10%), in the analyses of GSH in the lysates of human red blood cells and MCF-7 cancer breast cells in the presence and absence of the anti-inflammatory drug sulfasalazine. The internal-standard SALDI-MS approach provides simplicity, accuracy, and precision to the determination of GSH in cells under drug invasion, to open an avenue for SALDI-MS to be used for the precise quantitative determination of a variety of analytes. From the Clinical Editor: This paper reports the development of a surface assisted laser desorption/ionization mass spectrometry method to precisely determine aminothiols-cysteine (Cys), glutathione (GSH), and homocysteine (HCys) © 2010 Elsevier Inc. All rights reserved. Key words: Aminothiols; Drug therapy; Gold nanoparticles; Internal standard; Mass spectrometry Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool for the rapid quantification and identification of cellular proteins and tissue biomarkers. 1,2 Unfortunately, inhomogeneous crystallization of analytes within organic matrices always creates “sweet spots” in MALDI-MS analyses—with corresponding problems of poor accuracy and poor shot-to-shot and sample-to-sample reproducibility. In addition, because of the relatively high intensity of the matrix's background signals in the low-mass range (b500 Da), MALDI- MS is not popular for the detection of small solutes (e.g., aminothiols). 3 Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using nanoparticles (NPs) as matrices has been developed to overcome the problems associated with MALDI-MS. 4 Commonly used matrices in SALDI-MS include gold (Au), 5,6 silver, 7 silicon dioxide, 8 titanium dioxide, 9 and iron (II,III) oxide NPs. 10,11 Similar to the role fulfilled by organic matrices, the NPs absorb energy from the laser irradiation and transfer it efficiently to the analytes to induce their desorption and ionization. Moreover, with or without being bioconjugated with recognition molecules, the NPs can act as selective probes. Internal standards are commonly used in MS to improve the accuracy when determining the concentrations of analytes. The MS-based internal standards are often specifically isotopically tagged amino acids of the proteolytic peptides of interest. 12,13 For example, isotope-coded affinity tags comprising a thiol- reactive group, an isotope-coded linker, and a biotin affinity tag serve as cysteine (Cys)-labeling agents for the accurate quantification and sequence identification of individual proteins within complex mixtures. 13 An alternative approach is to add an internal standard (e.g., myoglobin) to the samples to improve the quantitative linearity and reduce the signal variations of MALDI- MS; this method has been used for the determination of disease- associated antigens. 11 Nevertheless, sample interference may be problematic when analyzing complicated matrices. In this study we developed a simple internal-standard method for improving the quantitative determination of the concentra- tions of aminothiols in cells through SALDI-MS using AuNPs. BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 530 – 537 Original Article www.nanomedjournal.com This study was supported by the National Science Council of Taiwan under contracts NSC 98-2627-M-002-013, NSC 97-2627-M-002-014, and NSC 98-2113-M-002-011-MY3. ⁎ Corresponding author: Department of Chemistry, National Taiwan University, Taipei 106, Taiwan. E-mail address: changht@ntu.edu.tw (H.-T. Chang). 1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2010.01.006 Please cite this article as: C.-K. Chiang, Y.-W. Lin, W.-T. Chen, H.-T. Chang, Research Article: EngineeringAccurate quantitation of glutathione in cell lysates through surface-assisted laser desorption/ionization mass spectrometry.... Nanomedicine: NBM 2010;6:530-537, doi:10.1016/j.nano.2010.01.006