Selective quantitative bioanalysis of proteins in biological fluids by on-line immunoaffinity chromatography-protein digestion-liquid chromatography-mass spectrometry.
A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography-mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 microl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.
Hoos
Johannes S
JS
Vrije Universiteit Amsterdam, Faculty of Sciences, Section Analytical Chemistry & Applied Spectroscopy, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. j.hoos@few.vu.nl
Sudergat
Hilke
H
Hoelck
Jens-Peter
JP
Stahl
Mark
M
de Vlieger
Jon S B
JS
Niessen
Wilfried M A
WM
Lingeman
Henk
H
Irth
Hubertus
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D002417:Cattle; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding / Q000276:immunology; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000276:immunology* / Q000652:urine
D000911:Antibodies, Monoclonal; D012710:Serum Albumin, Bovine