Use of an Immunoaffinity-Mass Spectrometry-based Approach for the Quantification of Protein Biomarkers from Serum Samples of Lung Cancer Patients
2008; Elsevier BV; Volume: 7; Issue: 10 Linguagem: Inglês
10.1074/mcp.m700476-mcp200
ISSN1535-9484
AutoresGordon R. Nicol, Mark Han, Jun Kim, Charles E. Birse, Erin Brand, Anh Nguyen, Mehdi Mesri, William W. Fitzhugh, Patrick Kaminker, Paul A. Moore, Steven M. Ruben, Tao He,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoIt 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. 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. 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Proteomics. 2007; 6: 2212-2229Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar) reported a work flow of optimized sample processing followed by MRM analysis for plasma proteins. Low ng/ml quantitation for several proteins in plasma was achieved. In the current study, an immunoaffinity-mass spectrometry-based approach was developed that uses antibodies to enrich multiple proteins of interest from sera followed by mass spectrometry-based quantification. Using this approach, we were able to observe CEA in serum for the first time by a mass spectrometry-based method, demonstrating the ability to quantify several potential protein biomarkers in sera in the low ng/ml range. We believe the immunoaffinity-MRM approach provides an attractive methodology to prioritize biomarkers for further assay development. Chemical reagents were obtained from Sigma except for mammalian protein extraction reagent and guanidine HCl (GuHCl), which were purchased from Pierce. Modified trypsin was purchased from Promega (Madison, WI). TIMP1, SLPI, TFPI, and TFPI2 antibodies and recombinant proteins were acquired from R&D Systems (Minneapolis, MN); CEA antibodies were purchased from Abcam (Cambridge, MA); and the recombinant protein was from United States Biological Inc. (Swampscott, MA). ELISA kits for TIMP1 and SLPI were purchased from R&D Systems, kits for CEA were from Biomeda (Foster City, CA), and kits for TFPI were from American Diagnostica (Stamford, CT). ELISAs were performed according to the suppliers' recommendations. Isotope-labeled peptides were purchased from Sigma-Genosys with either a heavy lysine (+8 Da) or heavy arginine (+10 Da). Lung cancer patient and age-matched normal sera were obtained from Clinical Research Center of Cape Cod (West Yarmouth, MA). Normal sera were purchased from Equitech-Bio (Kerryville, TX) and pooled for the spiking experiment. Where several antibodies were available, the antibody recommended by the manufacturer for immunoprecipitation or ELISA was chosen; otherwise polyclonal antibodies were preferred. Antibodies were immobilized on hydrazide beads (Bio-Rad, catalog number 153-6047) according to the manufacturer's protocol with minor modifications. Antibodies were dissolved or buffer-exchanged into DPBS. Carbohydrates on the antibodies were oxidized by incubating the antibody solution with NaIO4 (15 mm) for 1 h at room temperature in the dark with gentle shaking. After oxidation, 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. An aliquot was collected before and after binding for determination of binding efficiency by protein assay. After immobilization, the active sites on the resin were blocked with glyceraldehyde and washed several times with DPBS to remove any excess non-bound antibody. After determining the binding efficiency, the immobilized resins for all antibodies were combined and aliquoted such that ∼10 μg of each immobilized antibody was used for each sample. 100 μl of sera was diluted 10-fold with M-per (Pierce), and NaCl was added to a final concentration of 150 mm. Serum was incubated with the immobilized antibody resin overnight at 4 °C with gentle shaking. Unbound proteins were collected after incubation. The immobilized resin was washed three times with 1 ml of DPBS, and the protein was eluted with 2 × 100 μl 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 (∼2 pmol/peptide to monitor MS and LC performance from their individual m/z and elution profiles) were added to the eluted protein solution. Samples were reduced with DTT (5 mm) for 30 min at 60 °C and alkylated with iodoacetamide (20 mm) for 1 h at room temperature in the dark. Excess iodoacetamide was quenched by adding an additional aliquot of DTT. The sample was diluted to 0.8 m GuHCl with 0.1 m NH4HCO3, pH 8, and digested with trypsin overnight at 37 °C. The sample was desalted using a 1-ml Oasis hydrophilic-lipophilic balance cartridge (Waters, Milford, MA) and dried in a SpeedVac (Thermo-Savant, Waltham, MA). The samples were dissolved in 0.1% formic acid by gently vortexing for 30 s prior to LC-MRM analysis. 10% of the sample was injected on the 150-μm × 150-mm column, and 5% was loaded on the 75-μm × 150-mm column. LC-MRM was performed on an Agilent (Santa Clara, CA) 1100 LC system equipped with a 150-μm × 50-mm C18 Everest trap column and a 75-μm × 150-mm or a 150-μm × 150-mm C18 Everest analytical column (Vydac, Deerfield, IL). The analytes were loaded onto the trapping column and eluted using a gradient of 3–30% acetonitrile with 0.1% formic acid at a flow rate of 300 nl/min or 1.5 μl/min over 90 min. MRM data were collected on a 4000 Q TRAP instrument (Applied Biosystems, Foster City, CA) equipped with a NanoSprayII source. LC-MRM conditions were optimized for each peptide using the heavy isotope peptide standards. MRM transitions were acquired at unit resolution in both the Q1 and Q3 quadrupoles with 100-ms dwell time. 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. The large numbers of potential biomarker candidates being discovered from proteomics studies have overwhelmed current verification capabilities. Many of these potential candidates have no commercially available ELISA kits, resulting in no accurate way to quantify the protein levels in sera. Many candidates have antibodies available that have not been studied extensively for specificity and sensitivity and may not be suitable for ELISA measurements. Routine quantification of these biomarkers in sera is essential for the characterization of these candidates for diagnostic applications. Mass spectrometry is capable of quantifying proteins present in μg/ml and higher concentrations; however, many of the traditional biomarker candidates (e.g. CEA and prostate-specific antigen) are present in the ng/ml range. The complexity and the large dynamic range of proteins in sera make quantification in the ng/ml range by mass spectrometry a challenging task. Our approach was to use antibody affinity for the enrichment of the proteins of interest. We demonstrate that the use of immobilized antibodies to enrich the potential biomarker candidates followed by MRM mass spectrometry for quantification with stable isotope-labeled internal standards leads to the ability to quantify proteins in the ng/ml range in sera (Fig. 1). Antibodies for each protein were obtained from commercial sources, and immobilization was achieved as described under "Experimental Procedures." Binding efficiencies ranged from 60 to 95% and were taken into account when mixing the antibodies together for multiplexing so that ∼10 μg of antibody/protein was used in each experiment. Antibodies with lower specificities are acceptable in these experiments, increasing the number of antibodies available for this approach. Utilizing this approach takes advantage of the high affinity of the antibody to capture and therefore enrich antigens that are in very low abundance in sera and the extreme specificity of the MRM technology. The selection of the peptides used for quantification of the proteins of interest is critical. For candidates where recombinant protein was available, we digested the protein and determined the peptides that produced the best signal in an LC/MS experiment. Several peptides were chosen for each protein based on the intensity of the extracted ion chromatogram and their fragmentation pattern in MS/MS mode. The ideal peptide should have several (three to five) intense fragments (transitions) and a minimal number of less intense fragments. This need differs from the requirements of a peptide for identification purposes where more fragments provide greater confidence for data base matching. In contrast, for an MRM experiment, fewer peptide fragments result in a greater signal for each transition. The peptides chosen were analyzed in an MRM experiment with digested recombinant protein diluted into a background of digested sera. This enabled us to determine possible overlaps of transitions defined by the retention time, precursor m/z, and fragment m/z. Peptides with methionine residues were excluded from the list of candidates because of the possibility of oxidation during sample preparation. Post-translational modifications such as glycosylation interfere with digestion efficiency, and peptides containing these modification sites were excluded. All peptides were run through BLASTp (National Center for Biotechnology Information) to ensure that they were unique to the protein of interest. The peptide for each protein meeting these criteria and providing the most intense signal in the MRM experiment was selected for synthesis with heavy isotope incorporation. Table I shows a list of the peptides and transitions used for each protein analyzed. If the biomarker candidate did not have recombinant protein available, selection of peptides and transitions could be accomplished by using peptides observed in discovery experiments, identified from open source data bases (30Craig R. Cortens J.P. Beavis R.C. Open source system for analyzing, validating, and storing protein identification data.J. Proteome Res. 2004; 3: 1234-1242Crossref PubMed Scopus (574) Google Scholar, 31Deutsch E.W. Eng J.K. Zhang H. King N.L. Nesvizhskii A.I. Lin B. Lee H. Yi E.C. Ossola R. Aebersold R. Human Plasma PeptideAtlas.Proteomics. 2005; 5: 3497-3500Crossref PubMed Scopus (123) Google Scholar), or derived from the multiple reaction monitoring-initiated detection and sequencing (MIDAS) work flow (32Cox D.M. Zhong F. Du M. Duchoslav E. Sakuma T. McDermott J.C. Multiple reaction monitoring as a method for identifying protein posttranslational modifications.J. Biomol. Tech. 2005; 16: 83-90PubMed Google Scholar, 33Unwin R.D. Griffiths J.R. Leverentz M.K. Grallert A. Hagan I.M. Whetton A.D. Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity.Mol. Cell. Proteomics. 2005; 4: 1134-1144Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar).Table IPeptide sequences and transitionsProteinPeptidem/z precursorTransition selected fragment m/z of fragmentTFPIFFFNIFTR546.3y5 650.5y6 797.7y7 944.7SLPICLDPVDTPNPTR692.8y7 800.5y9 996.7y10 1111.6TFPI2YYYDRYTQSCR525.6y72+ 486y92+ 625y102+ 706.5CEACETQNPVSAR581.3y5 539.3y6 643.3y8 872.5TIMP1EPGLCTWQSLR673.9y6 790.7y7 950.4y9 1120.6aThe third transition for TIMP1 was not used for quantification due to interference for the heavy peptide.a The third transition for TIMP1 was not used for quantification due to interference for the heavy peptide. Open table in a new tab CEA was one of the first tumor-associated antigens identified and is expressed in nearly 50% of all human tumors (34Ballesta A.M. Molina R. Filella X. Jo J. Gimenez N. Carcinoembryonic antigen in staging and follow-up of patients with solid tumors.Tumour Biol. 1995; 16: 32-41Crossref PubMed Scopus (102) Google Scholar). In this study we examined the detection sensitivity of the immunoaffinity-MRM methodology for this known marker. CEA is a highly glycosylated protein with a molecular mass around 180 kDa. The molecular mass based on the primary amino acid sequence is 77 kDa, meaning that greater
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