title: Quantitative assay for six potential breast cancer biomarker peptides in human serum by liquid chromatography coupled to tandem mass spectrometry.
abstract: An assay to quantify several possible breast cancer peptide biomarkers in human serum has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The peptides include bradykinin, Hyp(3)-bradykinin, des-Arg(9)-bradykinin and fragments of fibrinogen alpha-chain (Fib-alpha([605-629])), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH(4[666-687])) and complement component 4a (C4a([1337-1350])). Ile(13)-ITIH(4[666-687]), d20-C4a([1337-1350]) and Sar-D-Phe(8)-des-Arg(9)-bradykinin were used as internal standards. Bovine plasma, with 2 mM captopril and 2 mM D-L-mercaptoethanol-3-guanidino-ethylthiopropanoic acid (MEGETPA) to prevent rapid degradation of the bradykinins, was used as analyte-free matrix. Recoveries for solid-phase extraction (SPE) on mixed-mode weak cation exchange sorbents were between 62 and 90%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with a heated electrospray source (H-ESI), operating in the positive ion-mode, was used for detection. The assay was fully validated and stabilities of the peptides were extensively explored. Bradykinin (10-500 ng/ml), Hyp(3)-bradykinin (4-200 ng/ml), des-Arg(9)-bradykinin (2-100 ng/ml), Fib-alpha([605-629]) (120-3000 ng/ml), ITIH(4[666-687]) (0.4-10 ng/ml) and C4a([1337-1350]) (1-25 ng/ml) were simultaneously quantified with deviations from the nominal concentrations below 22% and intra- and inter-assay precisions below 15 and 20%, respectively, for all peptides at all concentrations. The method has been successfully applied to several serum samples from breast cancer patients and matched controls.
authors.affiliation: Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Section of Biomedical Analysis, Division of Drug Toxicology, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. I.vandenbroek@uu.nl
authors.forename: Irene
authors.initials: I
authors.lastname: van den Broek
authors.forename: Rolf W
authors.initials: RW
authors.lastname: Sparidans
authors.forename: Jan H M
authors.initials: JH
authors.lastname: Schellens
authors.forename: Jos H
authors.initials: JH
authors.lastname: Beijnen
mesh_terms: D000510:Alpha-Globulins / Q000032:analysis; D000818:Animals; D014408:Biomarkers, Tumor / Q000097:blood*; D001920:Bradykinin / Q000031:analogs & derivatives / Q000097:blood; D001943:Breast Neoplasms / Q000175:diagnosis*; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D015934:Complement C4a / Q000032:analysis; D005260:Female; D005340:Fibrinogen / Q000032:analysis; D006801:Humans; D016014:Linear Models; D010949:Plasma / Q000378:metabolism; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000510:Alpha-Globulins; D014408:Biomarkers, Tumor; C464794:fibrinogen Aalpha; C011877:inter-alpha-inhibitor; D015934:Complement C4a; D005340:Fibrinogen; D001920:Bradykinin