Abstract
It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry-based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4-500 ng/ml), tissue factor pathway inhibitor (TFPI) (42-1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2-250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430-1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinity-multiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins for which the necessary reagents for ELISA development are unavailable.
Highlights
It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients
Reagents—Chemical reagents were obtained from Sigma except for mammalian protein extraction reagent and guanidine HCl (GuHCl), which were purchased from Pierce
The large numbers of potential biomarker candidates being discovered from proteomics studies have overwhelmed current verification capabilities
Summary
Reagents—Chemical reagents were obtained from Sigma except for mammalian protein extraction reagent and guanidine HCl (GuHCl), which were purchased from Pierce. Glycerol was added to quench the excess NaIO4, and the antibodies were desalted and buffer-exchanged into 0.1 M NaOAc, pH 5.5. Antibodies were incubated overnight with hydrazide beads at 4 °C with gentle shaking. Serum was incubated with the immobilized antibody resin overnight at 4 °C with gentle shaking. The immobilized resin was washed three times with 1 ml of DPBS, and the protein was eluted with 2 ϫ 100 l of 4.0 M GuHCl, 0.1 M Tris, pH 8. A standard mixture of heavy isotope-labeled peptides for the analytes (200 fmol/peptide) and 12 quality control peptides (ϳ2 pmol/ peptide to monitor MS and LC performance from their individual m/z and elution profiles) were added to the eluted protein solution. Quantification was accomplished using the Analyst QS 2.0 software package, and the areas of all analytes were normalized to the signals of their heavy stable isotope analogues
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