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Quantitative Proteome Analysis by Labeling of Arginine and Lysine with SILAC, SDS-PAGE, and nano-LC-MALDI-TOF/TOF Mass Spectrometry
| Type: | Application |
Application Note |
Expression Proteomics, Clinical Proteomics |
| Number: | Technology |
MT-86 |
MALDI-TOF |
| Year | Products |
2007 |
ultraflex II, WARP-LC, BioTools |
| Author | |
Frank Schmidt1, Arndt Asperger2, Detlev Suckau2, Bernd Thiede1 |
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| Reference | |
Bruker Daltonics Application Note MT-86 |
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Abstract |
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Quantitative proteome analysis of cisplatin-induced apoptosis in total Jurkat T cell lysates was performed in order to identify modified proteins. Proteins were labeled in cell culture with stable isotopes of arginine and lysine, fractionated by SDS-PAGE and analyzed by offline nano-liquid chromatography coupled to MALDI-TOF/TOF-MS. Data analysis was performed using the new WARP-LC 1.1 quantification software. Metabolic labeling of proteins with different isotopes (12C6 or 13C6, respectively) of arginine and lysine residues with subsequent tryptic digestion resulted in detection of all peptides as pairs in the mixture. Combined proteins derived from non-apoptotic and apoptotic cells revealed predominantly a heavy-to-light (H/L) ratio of about 1, indicating that most of the proteins were unchanged during apoptosis. Therefore, only about 2000 MS/MS spectra were acquired in total from the 60 gel slices due to the requirements to select peptides (H/L > 1.5 or L/H > 1.5) for MS/MS analysis although up to 1000 chromatographic compounds were detected per gel slice. As a result, several modified proteins with at least two peptide pairs were found by comparison of control and apoptotic Jurkat T cells. |
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