Quantitative assay for six potential breast cancer biomarker peptides in human serum by liquid chromatography coupled to tandem mass spectrometry.
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.
van den Broek
Irene
I
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
Sparidans
Rolf W
RW
Schellens
Jan H M
JH
Beijnen
Jos H
JH
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*
D000510:Alpha-Globulins; D014408:Biomarkers, Tumor; C464794:fibrinogen Aalpha; C011877:inter-alpha-inhibitor; D015934:Complement C4a; D005340:Fibrinogen; D001920:Bradykinin