Membrane Proteins in Bacterial Pathogenesis Laboratory Solution-­‐ICPMS Quantitation Solution ICPMS Inductively coupled plasma mass spectrometry (ICPMS) is a type of mass spectrometry that is highly sensitive and capable of the determination of a range of metals and several non-­‐metals at concentrations below one part in 1012 (part per trillion). It is based on coupling together inductively coupled plasma as a method of producing ions (ionization) with a mass spectrometer as a method of separating and detecting the ions. ICPMS is also capable of monitoring isotopic speciation for the ions of choice. Advantages: • Rapid Quantitative & Semi-­‐quantitative analysis of trace elements (6Li to 238U) • Multi-­‐Element Technique • Small Samples (< 100 mg) • High Sensitivity • Excellent Precision & Accuracy Disadvantages • Destructive Technique • Sample Homogenization • Sample Preparation • Spectral Interferences (many circumvented with Collision Cell) Solution ICP-­‐MS analysis requires the introduction of dilute solutions with total dissolved solids (TDS) less than 0.1%. All solutions must be filtered and free of particulates. Solutions should be diluted with 3.5% nitric acid. Standards A multi-­‐element standard is used which contains a broad assortment of metals (Mn, Fe, Zn, Cu, Ni). Fresh standards must always be prepared before each run. Working Stock = 1ml 10,000 ppb (10 ppm multi-­‐element standard) + 3ml 3.5% HNO3 = 2,500 ppb Standards Concentration Stock Diluent 500 ppb 1 ml Working Stock 4ml 3.5% HNO3 200 ppb 0.8 ml Working Stock 9.2 ml 3.5% HNO3 100 ppb 0.4 ml Working Stock 9.6ml 3.5% HNO3 50 ppb 1 ml 500 ppb 9 ml 3.5% HNO3 20 ppb 1 ml 200 ppb 9 ml 3.5% HNO3 10 ppb 1 ml 100 ppb 9 ml 3.5% HNO3 Membrane Proteins in Bacterial Pathogenesis Laboratory Sample Preparation Purified Protein Protein standards are prepared at 5µM and 10µM in duplicate, where possible. 1. Add the protein (x µl) to a final volume of 1ml of 3.5% HNO3. 2. Boil the samples at 95°C for 30 min taking care to cover the microcentrifuge tubes with another heating block. Use clip lock tubes (Eppendorf) to prevent popping of the lids. 3. Remove the insoluble material by centrifugation at 16,000 rpm for 20 min. 4. Collect the supernatant and dilute it with 1 ml of 3.5% HNO3 in a vial for analysis. 5. Store in the cupboard until analysis. Whole Cells Whole cells are harvested from small-­‐scale bacterial growth experiments (i.e. 50 ml cultures) in a tube of known mass (i.e. pre-­‐weighed) and dried overnight at 80°C. The dry cell weight of the cells can then be determined on a fine balance. 1. The dry cell pellet has 1 ml of 35% HNO3 added and then transferred to the heating block. 2. Boil the samples at 95°C for 30 min taking care to cover the microcentrifuge tubes with another heating block. Periodically (once or twice) carefully remove the tubes from the heating block, allow the tubes to cool briefly and vortex. Replace the tubes and continue to boil. 3. Remove the insoluble material by centrifugation at 16,000 rpm for 20 min. 4. Collect the supernatant and dilute as 200 µl with 1.8 ml of 3.5% HNO3 in a vial for analysis for a final volume of 2 ml. This is performed in technical replicates. 5. Store in the cupboard until analysis.