Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

The overall goal of this technique is to be able to quantify different metalloproteins in complex standards based on the use of known metalloprotein standards. This method can answer key questions about the role of metals in biology. For example, how does the metal status of a protein change in disease?

A key advantage of this technique is it allows you to measure the metal status of a protein in its native state. Demonstrating the technique will be Amber Lothian, a PhD student from my lab. To begin, homogenized tissue, or cell culture samples, by manual downs or sonication for five minutes using Tris buffered saline.

Clarify homogenates by centrifugation at 16, 000 times G, for five minutes. After collecting the resulting supernatant, normalize the total amount of protein by determining the protein concentrations as described in the text protocol. Warm up and tune the inductively coupled plasma maspectrometor, using the manufacturer's protocols.

Dilute the metalloprotein standards to enjoy the fall within the range of zero to 500 micrograms per liter, using one percent nitric acid. To achieve standards within this range, do not use dilutions exceeding one and 20. Set up the order of injections by first analyzing the seven calibration levels.

Concentrations ranging from zero to 500 micrograms per liter, followed by the metalloprotein standards. The seven calibration levels are zero, one, five, ten, 50, 100, and 500 micrograms per liter. Next, select elements of interest.

Here, use iron, copper, zinc, cobalt, and iodine. As they are the elements that the protein standards are bound to. Select the elements in the acquisition method by opening the element selector tab, and adding the elements and their respective isotope masses that are to be analyzed.

Then, perform sample analysis with the instrument. The instrument software automatically creates the calibration curves for each of the elements being analyzed. Use the calibration curves to determine the concentration of metal in the unknown samples.

From the bulk analysis results, generate the metalloprotein standards that include the three most abundant trace elements in Mammalian tissues using a mixture of copper, zinc superoxide dismutase, and ferritin. Purge the hyperformance quick chromatography, or HPLC pumps, with 200 millimolar ammonium nitrate buffer for five minutes at a flow rate of five milliliters per minute. Once the system is purged, set the flow rate at zero point one milliliters per minute.

And connect the side's exclusion column. Connect the opposite end of the column to the UV detector, set the measure absorbance at 280 nanometers, using peak tubing. Gradually increase the flow rate of the column in increments of zero point one milliliters per minute, until a final flow rate of zero point four milliliters per minute is reached.

While this is occurring, check the tubing connections to the column for any leaks. Monitor the pressure of this system to ensure it does not exceed the column requirements by observing the pressure trace on the chromatogram in the software. Leave the column to equilibrate at the required flow rate over five to 10 column volumes.

After it is equilibrated, connect the instruments to allow sample analysis to occur. Set up the HPLC method to pump buffer A over the column at zero point four milliliters per minute, for 15 minutes. Place the ICPMS into standby mode before changing the hardware setting.

Once in standby mode, turn off the communication for the auto sampler, and change the sample introduction to other. Use PEEK tubing to connect the outflow from the UV detector directly to the nebulizer on the ICPMS. Power up the ICPMS, and allow the instrument to warm up for 10 to 20 minutes.

After the plasma has ignited, connect the remote cable from the ICPMS to the back of the HPLC auto sampler. Do this once the plasma has ignited. Otherwise, the HPLC system will automatically shut down, since the ICPMS is not on.

Change the ICPMS method to collect data for LC ICPMS by first altering the acquisition method from spectrum to time resolve acquisition. Then, select the elements to be analyzed. Iron, copper, zinc, cobalt, iodine, as well as any other elements of interest, and set integration times.

After the elements of interest are chosen, adjust the acquisition time to match the run time for the chromatography. Manually tune the ICPMS for sensitivity and collision cell helium gas flow rates with a chromatography buffer flowing with cesium and antimony included in the buffer. Once the metal ion counts have stabilized, and relative standard deviation values are below five percent, the system is ready to use.

Make sample run lists in both the HPLC and ICPMS software programs. The sample run list contains the order in which each of the samples is to be injected. As well as the name by which the data will be saved.

Check that the total number of samples in the list match between the two programs. Start the sample batch for the ICPMS before the HPLC, as the injection of the sample by the HPLC will trigger the ICPMS to start collecting data. If this is not done in the correct order, it will result in missing data.

Generate calibration curve points by injecting varying volumes of the superoxide dismutase and ferritin mixed standard. Injection volumes range from one microliter to 30 microliters. Finally, analyze each of the unknown samples, such as tissue, plasma, or cell culture, before analyzing the data as described in the text protocol.

The illusion of ferritin over a range of 2000 to 60, 000 picograms of iron injected on the column is shown. Regression analysis was performed using peak area. The illusion profiles for copper, zinc, superoxide dismutase are shown for both copper and zinc.

The regression analyses generated using peak areas are also shown. The results of the regression analysis are used to convert the raw data in counts per second to picograms per second. So that the amount of metal associated with the protein can be determined quantitatively.

This technique can be used to identify metalloproteins in complex biological samples. Here, human brain separated by SEC ICPMS is shown. Each of which represent a different metal of interest.

Namely, copper, zinc, or iron. Shown here, are the traces obtained when human plasma is subjected to this technique. The complexity and abundance of the sample will impact the number of peaks that are seen.

As expected, plasma is dominated by a few metalloproteins, including ceruloplasmin and transferrin. After watching this video, you should have a good understanding of how to set up an instrument, to measure the native state of metalloproteins in complex samples. Once mastered, this technique can be successfully applied in three to four hours.

This allows for 20 to 36 samples to be analyzed in a single day. While attempting this procedure, it's important to use the appropriate metalloprotein standards, such as copper zinc SOD if you're interested in copper and zinc. As well as the internal standards in the buffer, such as cesium, which allows you to correct for any drift in instrument over time.

Following this procedure, you can also implement other forms of mass spectrometry to answer important questions. Such as, what are the metalloproteins present in my sample? This can be done by standard trips in digestion and mass spec protein peptide sequencing.

Or, by analyzing the proteins in tact, in their native state. After its development, this technique can pave the way for researchers in the field of proteomics to understand the role of metalloproteins in diseases, such as Alzheimer's disease. Don't forget that working with concentrated nitric acid, and clinical samples.

Appropriate personal protective gear should be worn.