Method Article
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.
Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.
Essential metals play a vital role in normal biological functions, including secondary messenger pathways, metabolism pathways and organelle functions. 30% of all proteins are thought to be metalloproteins1 and 50% of all enzymes2. Metalloenzymes use these trace metals as cofactors to catalyze chemical reactions, stabilize protein structure, and for regulatory roles such as secondary messengers. Some of the most studied trace elements in regards to neurodegeneration are copper, iron and zinc3. They are thought to be involved in many disease pathways where by dyshomeostasis can have adverse affects. For example the metal status of superoxide dismutase (SOD) directly impacts the life span and phenotype of the transgenic mice models of familial type of amyotrophic lateral sclerosis (ALS)4. In Alzheimer's disease, metalloproteomics techniques have been used to discover a decrease in the metal status of transferrin in plasma5. These studies highlight the important role metalloproteins can play in disease.
The study of metalloproteins directly from biological tissues is a developing field. Although some metalloenzymes have been characterized, the majority still remain uncharacterized or unknown6. One of the major challenges in measuring metalloproteins is the requirement to maintain the native state of the protein7. Classical bottom-up proteomic techniques rely on the digestion of the proteins into peptides. This process disrupts the non-covalent interaction of metals and their proteins. Thus, no information about the metal status of a protein is gained.
One way to overcome this issue is by using size exclusion chromatography paired with inductively coupled plasma - mass spectrometry8,9 (SEC-ICP-MS). This generates information about the protein's approximate size as well as any metals that are associated with it10. Further, size exclusion is a gentle chromatographic technique that can preserve the native state of an enzyme or protein-protein complex. One advantage of using inductively coupled plasma - mass spectrometry (ICP-MS) is the quantitative nature of the technology. Using a set of metalloproteins standards it is possible to provide absolute quantitation of metalloproteins from biological samples9,11. This is achieved by generating a standard curve by injecting known metalloproteins over a range of metal concentrations.
This protocol shows an example of how this can be achieved for a variety of metalloprotein standards. In this paper we aim to create standard curves for metals that are largely investigated in biological fields including iron (Fe), copper (Cu), zinc (Zn), iodine (I), and cobalt (Co).
1. Preparation of Buffers and Samples
2. Bulk Analysis of Metalloprotein Standards Using Inductively Coupled Plasma - Mass Spectrometry
3. HPLC System Setup and Size Exclusion Chromatography Column Equilibration
Note: This section should be performed in parallel to the previous, since they both required about 1 - 1.5 hr to complete.
4. Setting Up and Running Size Exclusion - Inductively Coupled Plasma - Mass Spectrometry
Note: Operating procedures may vary between instruments and models. Contact the instrument technical specialist to learn more about how to configure the ICP-MS being used.
5. Data Analysis, Manipulation and Visualization
The use of metalloprotein standards allows for the calibration of the size exclusion column. Figure 1A shows the elution profile for the standards thyroglobulin, ferritin, ceruloplasmin, Cu/Zn SOD and Vitamin B12 based on the metal that they are bound to (Fe, Co, Cu, Zn and I). Figure 1B shows the calibration curve for the size exclusion column based on the molecular weight of protein standards and their elution time, presented in the format of elution volume (Ve) divided by the void volume of the column (Vo). The proteins used to generate this standard curve are concanavalin A, conalbumin, ceruloplasmin, ferritin, SOD and thyroglobulin.
Figure 2A shows the elution of ferritin over a range of 2,000 - 60,000 pg of Fe injected on column and Figure 2D is the regression analysis performed using peak area. Figures 2B and 2C are the elution profiles for Cu/Zn SOD for Cu and Zn and 2E and 2F are the regression analyses generated using peak areas. The results of the regression analysis are used to convert the raw data in counts/sec to pg/sec so the amount of metal associated with the protein can be determined quantitatively. The conversion is done by dividing the counts/sec by the slope of the linear regression (e.g., 334.6 (counts/sec) x (sec/pg) of copper).
As stated, this technique can be used to identify metalloproteins in complex biological samples. Human brain and plasma have been subjected to this technique and Figures 3 and 4, respectively, show the results obtained. Human brain separated by SEC-ICP-MS is shown in Figures 3A-3C, each of which represent a different metal of interest (Cu, Zn or Fe). Figures 4A-4C show 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.
Figure 1. Calibration of Size Exclusion Chromatography - Inductively Coupled Plasma - Mass Spectrometry Using Known Metalloproteins. (A) Elution profile for the metalloprotein standards based on their respective metals. (B) Molecular weight calibration curve for protein standards thyroglobulin (i), ferritin (ii), ceruloplasmin (iii), conalbumin (iv), Cu/Zn SOD (v) and concanavalin A (vi). Please click here to view a larger version of this figure.
Figure 2. Use of Ferritin and Cu/Zn SOD as Metalloprotein Standards to Determine the Amount of Cu, Fe or Zn Associated with Metalloproteins in a Complex Biological Sample. (A) Elution profile for ferritin over the injection range 2,000 - 60,000 µg/L of iron. (B) and (C) Elution profile for Cu/Zn SOD over the injection range of 200 - 6,000 µg/L of copper and zinc, respectively. (D), (E) and (F) show the regression analysis results for the metals iron, copper and zinc, respectively. Please click here to view a larger version of this figure.
Figure 3. Cu, Fe and Zn Metalloproteome of Human Brain. (A) Copper trace (B) Iron trace (C) Zinc trace. Elution of the protein standards for each metal is shown by the black trace with their molecular weight indicated under the graph. Please click here to view a larger version of this figure.
Figure 4. Cu, Fe and Zn Metalloproteome for Human Plasma. (A) Copper trace (B) Iron trace (C) Zinc trace. Elution of the protein standards for each metal is shown by the black trace with their molecular weight indicated under the graph. Please click here to view a larger version of this figure.
Ensuring the native state of the protein means special attention to the buffers used and storage of the sample are needed. Not all chromatography techniques or sample preparation techniques can be employed. It is important that the buffers used throughout sample preparation and chromatography are devoid of metal chelators and use buffers that mimic physiological pH and salt concentrations. Other conditions to avoid include heating the sample or addition of protein denaturants (e.g., urea). It is critical to minimize the number of freeze thaw cycles. The ability of the chosen buffer to bind divalent metals is also important and is a reason that Tris or ammonium nitrate buffers are chosen over phosphate based buffers.
The low resolution and peak capacity of the size exclusion chromatography relative to other forms of chromatography is a major limitation of this technique. However, the gentle nature of size exclusion chromatography is important to maintain the native state of the protein and thus preserve the relatively weak metal-protein bonds. The requirement to maintain the native state of the proteins requires special attention to the treatment of the samples including limiting the number of freeze thaw cycles, avoiding metal chelators (e.g., EDTA) or chaotropic salts and detergents.
This low resolution of this technique impacts the ability to quantify the amount of metal associated with a specific protein if it is in a complex sample as the peaks seen will contain more then one protein. Therefore, the amount of metal determined using the peak integration would be an indication of the total amount of metal associated with all of the proteins eluting at this time point and not just one specific protein. In order to overcome this limitation the protein of interest would have to be further purified under native conditions. This would allow for the quantification of the metal associated with this protein to be reported with a higher degree of certainity. Another potential limitation of this technique would be the loss of protein due to non reversible binding to the column. In order to determine if this is happening a recovery experiment should be carried out whereby the amount of protein eluting off the column is analysed to determine if this matches the amount injected. The same can be done by measuring the metal content of the elution material and the starting material by bulk ICP-MS. Column recovery can differ depending on the conditions used, but it has been shown that complete recovery of proteins from a size exclusion column is possible9. Thus it is important to check whether or not there is any loss under the operating conditions being used.
Modifications to the protocol can be related to the metalloprotein standards that are used as well as the elements analyzed. The type of metalloprotein standard used will differ depending on the elements that are of interest. For elements such as Cu, Fe and Zn proteins, SOD and ferritin are employed. Any other metalloprotein that have known stoichometries can also be used and a few examples have been shown here.
One major complication that can arise from using this technique is the build up of salt crystals in the torch of the ICP-MS. To prevent the build-up of salt crystals, the torch is washed with distilled water after every 500 - 1,000 ml of buffer that has been passed through the system or when it is determined by visual inspection that the torch should be washed. Another problem that can arise is a more rapid decline in the cleanliness of the sample and extraction cones. These need to be cleaned regularly following manufacturer's protocols.
The initial sample preparation is the most critical step in the protocol. If there are any changes to the protein - metal complex the information generated will not be valid. This is one of the major limitations of the technique; in addition the use of the low resolution, size exclusion column yields a limited detailed view of the true complexity of metalloproteins in biology.
The technique described here allows the expansion of knowledge of the metalloproteome of an organism. Bulk analysis only gives a crude indication of changes to the amount of metal within a sample. Besides the general considerations that need to be taken into account, this technique provides a tool that can be used to quantitate the amount of metal associated with proteins as well as identifying metalloproteins that differ by comparing the traces obtained. The use of this technique can be employed to identify differences between disease states. The identified metalloproteins can then be further investigated to help determine the role they play in disease processes. The application of hyphenated ICP-MS has a growing future to determine the role of drugs that have a heteroatom such as platinum, iodine or copper as the ICP-MS can be used to identify the proteins that the drug binds.
The authors have nothing to disclose
We would like to acknowledge support from Victorian Government's Operational Infrastructure Support Program, the Australian Research Council Linkage Projects Scheme (with Agilent Technologies), the Australian National Health and Medical Research Council, the Victorian brain bank, Cooperative Research Centre for Mental Health and the Neuroproteomics facility.
Name | Company | Catalog Number | Comments |
Agilent 1290 Infinity Binary Pump | Agilent | G4220A | |
Agilent 1290 Infinity Autosampler | Agilent | G4226A | |
Agilent 1200 Series Autosampler Thermostat | Agilent | G1330B | |
Agilent 1290 Infinity Thermostatted Column Compartment | Agilent | G1316C | |
Agilent 1290 Infinity Variable Wavelength Detector | Agilent | G1314E | |
Agilent 7700 ICP-MS | Agilent | G3282A | |
Ammonium hydroxide trace metal basis | Sigma | 338818 | |
Ammonium nitrate | Sigma | 256064 | Make fresh 200mM solution on day of experiment |
Antinomy | Choice analytical | 10002-3 | |
Ceruloplasmin | Sigma | ||
Cesium | Choice analytical | 100011-1 | |
Complete, EDTA free protease inhibitors | Roche | 11873580001 | |
Conalbumin | Sigma | C7786 | |
Concanavalin A from Canavalia ensiformis (Jack bean) | Sigma | L7647 | |
Cu, Zn Superoxide dismutase | Sigma | S9697 | |
Ferritin | Sigma | F4503 | |
ICP-MS multielemental calibration standards | AccuStandard | Made up to required concentrations in 1% nitric acid | |
Microvolume UV spectrophotometer | Thermo Scientific | ||
65% Nitric acid | Millipore | 100441 | Diluted to 1% for use |
Peek tubing | Agilent | 5042-6461 | |
Size exclusion column BioSEC-3 PLC. column, 4.6 x 300 mm, 3 μm, 150 Å | Agilent | 5190-2508 | |
Sodium Chloride | Chem Supply | SA046 | |
Tris Hydrochloride | ICN Biomedicals inc. | 103130 | |
Thyroglobulin | Sigma | T9145 | |
Vitamin B12 | Sigma | V2876 |
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