Multiple reaction monitoring cubed for protein quantification at the low nanogram/milliliter level in nondepleted human serum.
Mass spectrometry-based strategies for the quantification of low-abundance putative protein biomarkers in human blood currently require extensive sample fractionation steps which hamper their implementation in a routine and robust way across clinical laboratories. We demonstrate that a technique using MS(3) reconstructed chromatograms on a signature of secondary ions issued from a trapped primary product ion, termed multiple reaction monitoring cubed (MRM(3)), enables targeting protein biomarkers in the low nanogram/milliliter range in nondepleted human serum. The simple two-step workflow is based on a trypsin proteolysis of whole serum (100 microL) followed by enrichment of targeted proteotypic peptides on a solid phase extraction column using mixed-cation exchange resin. MRM(3)'s fidelity of peak detection extends the dynamic range and limit of quantitation (LOQ) of protein biomarkers to the low nanogram/milliliter range, corresponding to a concentration that is 10(6)-fold lower than the concentration of the most abundant proteins in serum. The power of the MRM(3) method is illustrated by the assay of prostate specific antigen in nondepleted human sera of patients. The results correlate well with the established method for determining PSA levels in serum, i.e., enzyme-linked immunosorbent assay (ELISA) tests.
R&D Proteomique, bioMerieux SA, Marcy l'Etoile, France.
T
T
Fortin
A
A
Salvador
J P
JP
Charrier
C
C
Lenz
F
F
Bettsworth
X
X
Lacoux
G
G
Choquet-Kastylevsky
J
J
Lemoine
D014408:Biomarkers, Tumor / Q000097:blood*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D017430:Prostate-Specific Antigen / Q000097:blood*; D011471:Prostatic Neoplasms / Q000097:blood*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D014408:Biomarkers, Tumor; D017430:Prostate-Specific Antigen