Detection of metals and metalloproteins in the plasma of stroke patients by mass spectrometry methods

Abstract

Stroke is the leading cause of adult disability, worldwide. Metalloproteins and metals play key roles in epigenetic events in living organisms, including hypertension, the most important modifiable risk factor for stroke. Thus, metalloproteins may be important target biomarkers for disease diagnosis. The primary goal of this study was to assess metal containing proteins in blood plasma, detected by ICP-MS, followed by ESIMS for peptide/protein identification. We then compared the relative concentration differences between samples from patients with ischemic stroke, hemorrhagic stroke and stroke mimics. In 29 plasma samples (10 stroke mimics, 10 ischemic stroke and 9 hemorrhagic stroke patients) previously collected from patients who presented to the University of Cincinnati Emergency Department within 12 hours of symptom onset for a plasma banking project. For the metal associated protein study, Mg, Mn, Cu, Se concentrations were statistically different when compared between stroke mimics vs. ischemic stroke patients and ischemic stroke patients vs. hemorrhagic stroke patients. Pb concentrations were statistically different when compared between stroke mimics vs. ischemic stroke patients and Mo levels were statistically the same among the three groups. In addition, we also report concentration levels and preliminary correlation studies for total elemental analysis among the three sets of patients. This pilot study demonstrates that mass spectrometry methods may be highly valuable in detecting novel stroke biomarkers in blood plasma. Expanded studies are warranted to confirm these findings.

Figure 1

Chromatogram obtained from the immuno depletion of HSA and IgG from the plasma samples, the fraction collected for further separations was between 1.5 and 7.5 minutes for each sample. The fraction at 16 minutes is the HSA and IgG, which were separated from plasma samples after immuno-depletion (Affinity column).

Figure 2

Typical SEC-ICPMS chromatogram, as a second LC separation. The metal peaks shown above are as follows: a) Mg (Black trace) Cu (Blue trace), Pb (Red trace); b) Mn (Red trace) and Zn (Black trace); c) Al (Pink trace) and Mo (Blue trace) and d) Se containing protein (Blue trace).

Figure 3

Box charts obtained for all the metalloproteins. Charts obtained from Origin pro software, which describes the statistical difference between the three sets of stroke patients (Control, Hemorrhagic and Ischemic stroke). Box chart are graphical representations of key values from summary statistics and is helpful in quality analysis for rapid initial interpretion of the data distribution (Minimum 25^th percentile; median; 75^th percentile maximum).

Figure 4

Peptide mapping approach used to assign the protein IDs. a) Total ion chromatogram obtained by nanoLC-ESI-MS/MS, for one of the peaks in the reverse phase separation, after tryptic digestion and identified by MASCOT as Apolipoprotein A-I. Apolipoprotein A-I protein is obtained from SEC fraction 1. b) Extracted ion chromatogram at m/z 701.5 ± 0.3, shows the signal for the peptide DYVSQFEGSALGK as [M-H]²⁺ ion with good chromatographic behavior. c) MS fragmentation of the ion m/z 701.5. d) MS/MS fragmentation of the ion m/z 701.5.