Developing an LC-MS/MS method for hepcidin quantitative analysis

Transcription

1 Developing an LC-MS/MS method for hepcidin quantitative analysis Asian Pacific Conference on Chromatography & Mass Spectrometry January 2010 Alan L. Rockwood and David K. Crockett ARUP Laboratories, Dept. of Pathology, University of Utah School of Medicine

2 Introduction 25 amino acid peptide discovered in 2000 Produced in the liver, heart and brain Key regulator of iron homeostasis Affected by hypoxia and inflammation

3 History Adermann first described as LEAP Ganz reported "hep (from liver) cidin (antibacterial) Kaplan described interaction with ferroportin (iron regulation) Swinkels et al. many papers

4 Biochemistry Found in human urine and blood 3 isoforms: 20, 22, 25 amino acid Active form is 25 Eight cysteine residues - 4 internal S-S bonds Proper folding required for bioactivity Binds directly to ferroportin degradation Regulates (inhibits) iron transport into cells

5 NMR structure of hepcidin

6 Hepcidin function

7 Disease Anemia Anemia of chronic disease/inflammation Iron overload Juvenile hemochromatosis Immune function

8 Published analytical methods SELDI-TOF MS, Kemna et al, Blood 2005 LC-MS/MS, Murphy et al, Blood 2007 Immunoassay, Ganz et al, Blood 2008 Competitive binding (ferroportin)/fluorescence, De Domenico et al, Cell Metab 2008

9 Disagreement between methods For both urine and plasma the absolute hepcidin concentrations differed widely between methods Kroot JJ, et al. Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization. Haematologica Dec;94(12):

10 Analytical approach LC-MS/MS Sample Preparation Chromatography MRM quantitation

11 Why LC-MS/MS? Multiple forms of hepcidin Separation (bioactive vs. others) Better quantitative analysis potential More stable ionization Separation

12 Sample preparation methods tried Solid phase extraction Spin filter (10 kda cutoff) Protein precipitation microliter Several internal standards Urine and serum together

13 Sample prep comparison Summary of hepcidin recovery by solid phase extraction, spin filter, and precipitation. Method 0.5 ng/ml 5.0 ng/ml 50.0 ng/ml Cost per sample Control spiked a 100% 100% 100% -- SPE - Strata X a 79% 81% 87% $1.10 SPE - Oasis HLB a 84% 89% 81% $1.40 SPIN - Princeton (Centri-Spin20) b 3.9% 3.4% 4.8% $1.25 SPIN - Pall (AcA 202) b 5.6% 6.4% 6.7% $0.92 SPIN - Millipore (YM-10) b 86% 85% 89% $2.78 Acetonitrile precipitation b 101% 97% 108% $0.01 a Average recovery calculated from n=3 replicates. b Average recovery calculated from n=5 replicates.

14 Optimized sample preparation 100 ul patient serum/urine Acidify with 1% formic acid Spike with internal standard Acetonitrile precipitation or size filtration Internal standard: 25-mer related peptide

15 Internal standard example Heavy isotope labeled hepcidin 25 DTHFPI*CI*FCCGCCHRSKCGMCCKT * two heavy isoleucine residues (C13, N15) Incorporated mass difference = 16 Oxidized methionine (16 dalton) may interfere

16 Internal standard options 1. Hepcidin Hepcidin 25 related sequence 3. Unrelated human 19-mer heavy isotope labeled 4. Hepcidin 25 heavy isotope labeled 4 x ESI hep25_is_summary.d Counts vs. Acquisition Time (min) Q-TOF data

17 Inline enrichment chromatography (quasi-2-d) Enrichment Phenomenex C18 (4mm L x 2mm ID) Analytical Phenomenex C18 (50mm L x 2mm ID) A Pump A B Pump A (A) Loading Analytical column Autosampler Analytical column Autosampler (B) Analysis to MS Trap Pump B to MS Trap Pump B Waste Waste

18 Mass spectrometers used AB/Sciex API 4000 (QQQ) Agilent 6410 (QQQ) Agilent 6510 (Q-TOF)

19 Instrument comparison Summary of optimized hepcidin 20, 22, and 25 m/z, charge state, collision energy and fragment ions. charge state (z) collision energy dominant peptide MW * QTOF QTrap transition hepcidin hepcidin 20 heavy a hepcidin hepcidin 22 heavy a hepcidin hepcidin 25 heavy a a incorporates two isoleucine (13C, 15N) isotope labeled resides. dominant charge state on Agilent 6410 QQQ. dominant charge on Agilent 6510 QTOF. * dominant charge state on API 4000 QTrap. Optimum ions and MS/MS transitions depend on choice of instrument type

20 Calibration Hepcidin 25 calibrators Hepcidin 25 calibrators y = x Four point 1.20 curve (1, 2, 5, 10 ng/ml) in y = x serum R 2 R 2 = = Analyte Area/IS Area Analyte Area/IS Area Analyte Conc./IS Conc. Analyte Conc./IS Conc. 200 microliter sample volume API 4000 instrument

21 Accuracy and Precision Sample (ng/ml) a Intra-assay CV% Inter-assay CV% ACC% % 13.2% 105.0% % 6.7% 98.8% 0.5 b 5.7% 6.4% 96.5% % 6.2% 92.5% % 1.7% 99.0% % 3.7% 109.3% % 5.9% 95.1% c 3.2% 2.1% 104.3% a Limit of detection b Limit of quantitation c Upper limit of linearity 200 microliter sample size API 4000 instrument

22 Reference interval 300 volunteers total Matched urine and serum samples 60 excluded from reference intervals (due to abnormal ferritin concentration) 240 healthy individuals based on serum ferritin concentration 120 male 120 female

23 Mass spectrometer for reference interval determinations API 4000 QTrap (Applied Biosystems/Sciex) Turbo-spray ESI source MRM acquisition hepcidin 25 (z = +4 and +5 parent ions) internal standard (z = +4 and +5 parent ions)

24 Nonparametric reference interval in serum (ng/ml) Male Female Mean = Median = 6.30 N = 120 CI = 1.01 to Mean = 9.08 Median = 6.47 N = 120 CI = 0.90 to 31.61

25 Nonparametric reference interval in urine (ng hep25/mg Crt)* Male Female Mean = Median = 7.00 N = 120 CI = 0.39 to * Urine hepcidin expressed as a ratio to creatinine Mean = Median = N = 120 CI = 0.11 to 61.51

26 Bioactive Hepcidin-25 QTOF analysis of bound fractions from ferroportin peptide binding assays was performed. Human serum was incubated with the hepcidin binding domain peptide from beads. Crockett DK, Kushnir MM, Phillips JD, Rockwood AL. Time-of-flight analysis of the ferroportin-hepcidin binding domain complex for accurate mass confirmation of bioactive hepcidin 25. Clinica Chimica Acta. Epub 2009 Dec 3. De Domenico, et al. Cell Metab Aug;8(2):

27 Confirmed MRM masses (A) 5 x BPC Scan hep25_2d_flow through.d 1 1 Flow through Counts v s. Acquisition Time (min) TOF analysis of binding assay fractions. (B) 7 x TIC Scan hep25_3d_bound.d Bound Counts v s. Acquisition Time (min) Confirms our MRM measurement of bioactive hep25.

28 Subtleties and pitfalls Extraction and storage losses (adsorption, freeze/thaw, etc.) Correct folding/purity of standards Multiple forms (isobaric and non-isobaric) Targeting correct (bioactive) form

29 Subtleties and pitfalls Temperature effects (conformation?) Proper choice of internal standard Method optimization different between instruments Correlation between methods

30 Conclusions LC-MS/MS method for hepcidin developed Reference intervals determined Differences in instrument optimization between instruments Pitfalls must be recognized and avoided

31 Acknowledgments ARUP Laboratories ARUP Institute for Clinical and Experimental Pathology, Department of Pathology, University of Utah School of Medicine Reza Panahi Graduate Student, Clinical Laboratory Science Program, Dept. of Pathology, University of Utah School of Medicine John D. Phillips, PhD Internal Medicine, Hematology, University of Utah School of Medicine