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Blood, Vol. 110, Issue 3, 1048-1054, August 1, 2007
Quantitation of hepcidin from human and mouse serum using liquid chromatography tandem mass spectrometry
Blood Murphy et al.
110: 1048
Supplemental materials for Murphy et al, Vol. 110, Issue 3, 1048-1054
Files in this Data Supplement:
- Document 1. Supplemental method: infusion of hepcidin standard solutions (PDF, 10.2 KB)
- Document 2. Supplemental method: LC/MS/MS of hepcidin in plasma and serum samples (PDF, 9.80 KB)
- Figure S1. Full and product ion mass spectra of human and mouse hepcidin synthetic standards (PDF, 18 KB) -
(A) Full scan (300 – 1500 m/z) spectra show ions of human hepcidin at m/z 698.4 (M+4H)+4 and m/z 558.8 (M+5H)+5. (B) Product ion scans of the m/z 698.4 (M+4H)+4 ion show the product of m/z 644.1 (y23)+4 as the most abundant fragment ion for human hepcidin. (C) The full scan spectra of mouse hepcidin shows ions of m/z 919.1 (M+3H)+3 and m/z 689.1 (M+4H)+4. (D) Product ion scans of the m/z 919.1 (M+3H)+3 ion show the product ion of m/z 1139.4 (y21)+2 as the most abundant fragment ion for mouse hepcidin.
- Figure S2. Extracted ion traces for human hepcidin in rabbit and human serum (PDF, 14.6 KB) -
Extracted ion traces are shown for the human hepcidin SRM (m/z 698.4 > 644.1) in (A) control rabbit serum and (B) a 1 ng human hepcidin/mL rabbit serum . The ion trace intensity for hepcidin in Figure S2A is normalized to the hepcidin response shown in Figure S2B. Extracted ion traces are also shown for (C) normal pooled human serum and (D) the same pooled human serum with a standard addition of 5 ng/mL human hepcidin. No detectable hepcidin levels were observed from the pooled human serum. The ion trace intensity for hepcidin in Figure S2C is normalized to the hepcidin response shown in Figure S2D. Internal standard extracted ion traces (SRM m/z 758.9 > 711.7) are also shown for each sample. Retention times for human hepcidin and the internal standard varied by less than 0.04 minutes within each sample batch.
- Figure S3. Extracted ion traces for mouse hepcidin in rabbit and mouse serum (PDF, 14.4 KB) -
Extracted ion traces are shown for mouse hepcidin (m/z 919.1 > 1139.4) in (A) control rabbit serum and (B) a 0.25 ng mouse hepcidin/mL rabbit serum. The ion trace intensity for hepcidin in Figure S3A is normalized to the hepcidin response shown in Figure S3B. Extracted ion traces also shown for (C) normal pooled mouse serum and (D) the same pooled mouse serum with a standard addition of 20 ng/mL mouse hepcidin. The pooled mouse serum yielded an average calculated concentration of 3.15 ng/mL hepcidin before standard addition. The ion trace intensity for hepcidin in Figure S3C is normalized to the hepcidin response shown in Figure S3D. Internal standard extracted ion traces (m/z 758.9 > 711.7) are also shown for each sample. Retention times for mouse hepcidin varied by less than 0.04 minutes within each sample batch. The mouse diet used for this study was certified rodent diet 5002 with reported iron levels of 210 ppm.
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