Reducing Sample Amounts for Isobaric Tagging Quantitative Proteomics Experiments

• Published in: Journal of biomolecular techniques 2014-05, Vol.25 (Suppl), p.S31-S31
• Main Authors: Loprest, M., Colangelo, C. M., Abbott, T. B.
• Format: Article
• Language: English
• Subjects: Poster Abstracts
• ISSN: 1524-0215; 1943-4731

Quantitative proteomics by mass spectrometry relies on the accurate comparison between multiple samples, using several methods including isobaric tagging reagents such as iTRAQ or TMT. These reagents enable multiplexing where the area of the reporter ions present in the MS/MS spectrum allows comparison across samples within one run. However, the cost of the isobaric tagging kits deters many researchers. In addition, the iTRAQ labeling reaction is dependent upon both concentration and absolute amount of sample present, and supplier documentation recommends reactions on 20 to 100 micrograms of protein. Since the current generation of high resolution mass spectrometers only need a few micrograms of protein for analysis, developing methods to utilize the isobaric reagents to label multiple sample sets at lower sample amounts would represent a significant savings per experiment. To test this theory, we utilized a single iTRAQ kit to label multiple sets of a quantified HEK 293 tryptic digest at 10, 5, and 1 ug amounts. In order to effectively label 10 ug, 5 ug, and 1ug, we utilized 16.3%, 8.1%, and 1.7% of the reagents in 17, 8.5, and 1.7 ul volume. Differing percentages of each labeled sample were chosen to yield measureable iTRAQ ratios when mixed. Samples were desalted on C18 to remove undigested protein and excess iTRAQ reagent, dried, and redissolved in 70% formic acid, 0.1% trifluoracetic acid in water, and run on an AB Sciex 5600 Triple TOF mass spectrometer. Preliminary results show efficient iTRAQ labeling for all concentrations. Additionally, the 10 and 5 ug loads showed minimal loss of protein identifications when compared to 20 ug experiments, while 1 microgram showed some loss due to inefficient sample recovery at the C18 step. TMT labeling reactions using similar conditions are currently in progress and will also be reported.