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33


Gel marker filtration kit and myoglobin stored at -20°C, all others stored at 4°C.


Chemicals and Reagents:- NaCl (laboratory reagent), Sodium phosphate monobasic (NaH2 (Na2


PO4 HPO4


), Sodium diphosphate dibasic ), Fe, Cu and Zn 1000 ppm


standards (in nitric acid) were all obtained from Fisher Scientific, UK.


SEC-HPLC Separation


The SEC-HPLC analysis was carried out [4] using a Perkin Elmer HPLC system composed of a series 200 pump, series 200 vacuum degasser and series 200 UV/ Vis detector with a 20 µL injection loop. The chromatographic column used was the TricornTM


temperature does not affect retention times. Figure 2 shows the 4 metal containing proteins used in this investigation.


ICP-MS analysis


Analysis of all samples and standards [5] was performed on a Perkin Elmer NexIon 350X ICP-MS, in KED mode, tuned using NexIon Setup Solution (Perkin Elmer, UK). Fe, Cu and Zn standards were produced and diluted in 1% HNO3


to the following


concentrations: 0, 0.05, 0.1, 0.5, 1 ppm. SEC-HPLC-ICPMS Analysis


Superdex 200 10/300 GL high


performance column (GE Healthcare; bed dimensions; 10 x 300 -310 mm, column volume 24 mL; particle size 13 µm).


The column was first calibrated using gel marker filtration kit (mw 12,000 – 200,000 Da) which is a standard protocol for SEC separation. The column was further calibrated with 4 metal containing proteins: haemoglobin, myoglobin, transferrin and ferritin. SEC-HPLC analysis was performed under the following conditions. Flow rate 0.5 mL/min, mobile phase composed of 50 mM Na2


HPO4 •2H2 O and 0.15 M NaCl


adjusted to pH 7.2 with HCl. Proteins were detected by UV/Vis detector at 280 nm. Column pressure was between 120-130 psi to allow for additional back pressure in the system (column max 217 psi). Ambient lab temperatures were recorded daily (as is common practice by students working in our laboratories), temperatures ranged from 21-29°C throughout the course of analysis, however it should be noted that SEC systems operate in such a way that


SEC-HPLC was hyphenated to the ICP- MS instrument previously discussed using the same conditions, using Syngistix and Chromera software. Protein standards, blood extracts and control samples were analysed.


Results & Discussion


This section is composed of results acquired during method development procedures as well as genuine investigative results.


SEC-HPLC Method Development Gel Filtration Marker Kit Calibration


Conditions initially used for SEC-HPLC analysis were 50 mM phosphate buffer + 0.15M NaCl at pH 7.2 with a flow rate of 0.5 mL/min and a run time of 180 minutes, as the time for protein separation was unknown under these specific conditions. To test the separation capabilities of the column and gauge the separation time of the proteins, proteins from a gel marker filtration kit (mw 12,000 – 200,000 Da, MWGF200 Sigma) were separated on the column. Duplicate runs were performed for all protein separations in this analysis.


The smallest molecular weight protein in the kit, Cytochrome C, was analysed first to approximate how long run times would be. Following this the largest molecular weight protein was run followed by all other proteins to determine each individual retention time. In this case, the void volume was calculated using blue dextran. This was prepared to a concentration of 1 mg/mL. A combined mixture of standards was also prepared and run.


Metal-containing Proteins Calibration


Figure 2. From bottom to top, chromatograms of Haemoglobin, Myoglobin, Transferrin and Ferritin.


All protein standards were first run separately in duplicate to determine individual retention times. Set concentrations were used for proteins as a means to produce peaks of similar height. The concentrations of the myoglobin, transferrin and haemoglobin standards were 3 mg/mL, while the standard of ferritin, which has a higher


rate of absorbance, was prepared at a concentration of 0.3 mg/mL. Myoglobin and Cytochrome C are close in molecular weight and could not be separated under these conditions.


Dried Blood Spot Extraction Method Development


Blood spots were extracted under varying conditions of time and temperature as well as in different solutions to determine an optimal procedure. However, all early trial methods gave satisfactory extraction results and therefore, all subsequent extractions were done in 2 mL of water at 40°C for 60 minutes. All control samples were subject to the same conditions.


DBS SEC-HPLC


The SEC-HPLC conditions described in the standard analysis were utilised for this analysis of the DBS samples. Duplicate runs were done for all separations in this stage.


All dried blood spot samples extracted were separated via SEC-HPLC to determine the metalloproteins present and also to determine if the varying extraction conditions had an effect on separation results. From the retention times of the proteins separated in the samples, the proteins within the blood samples were identified as transferrin and haemoglobin. It was observed that the different extraction conditions did not affect the results of separation in the DBS samples. All samples show the same two proteins at similar retention times. Figure 3 illustrates these chromatograms. The two peaks present in the ferritin chromatogram can be attributed to heavy and light forms of ferritin being simultaneously present in the sample. Light and heavy ferritin have molecular weights of 19 kDa and 21 kDa respectively.


Figure 3. from left to right shows the SEC-HPLC chromatograms for Myoglobin, Haemoglobin, Transferrin, Ferritin and an extracted blood sample. Retention times of characteristic peaks were 33.09, 30.19, 26.34, 14.93 and 19.61, 29.93 minutes respectively.


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