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LC-MS/MS quantification of Zn-[alpha]2 glycoprotein: a potential serum biomarker for prostate cancer.

Zn-[alpha]2-glycoprotein (ZAG) [1] is a glycoprotein with a molecular mass of ~41000 Da and a crystal structure similar to that of a class I major histocompatibility complex (1,2). Biochemically, ZAG stimulates lipid degeneration in adipocytes and appears to be involved in cachexia, a wasting syndrome that can affect people with cancer, AIDS, and other terminal illnesses (3,4). ZAG appears naturally in most body fluids, such as blood (5), sweat (6), seminal fluid (7), breast cyst fluid (8), cerebrospinal fluid (9), and urine (10) and is also found in secretory epithelial cells of the liver and the gastrointestinal tract (11).

Previous studies employing techniques such as immunohistology and 2-dimensional electrophoresis have reported that ZAG is overexpressed in certain malignant tumors and thus may serve as a potential cancer biomarker (12,13). ZAG quantification in serum by immunoassay found circulating concentrations ranging from 40 mg/L in healthy individuals to 120 mg/L in some diseased persons (14). In this study we used liquid chromatography-tandem mass spectrometry (LC-MS/MS), at a high LC flow rate commonly used in the clinical laboratory (250 [micro]L/min), combined with proteolysis, to quantify ZAG in serum. We also determined whether ZAG could be used as a specific biomarker for prostate cancer (PCa).

Materials and Methods

RECOMBINANT ZAG PROTEIN STANDARD

The recombinant ZAG protein used in this study was a gift from Dr. Bjorkman (Division of Biology, Howard Hughes Medical Institute, California Institute of Technology). The protein was generated in CHO cells by transfection with a ZAG vector using Lipofectamine 2000 (Invitrogen) (2). The recombinant ZAG stock concentration was found to be 1.2 g/L as determined by BCA total protein analysis (Pierce). The purity of the protein was estimated to be >95% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1A, inset). Standard curves were generated by diluting the recombinant ZAG into 5% bovine serum albumin (BSA) in phosphate-buffered saline (10 mmol sodium phosphate, 150 mmol sodium chloride, pH 7.2).

SERUM DIGESTION

All digests were performed on 125 [micro]L aliquots of serum. No prior purification or removal of high-abundance proteins such as serum albumin was performed. Samples were first denatured with 6 mol/L urea and followed by reduction in 15 mmol/L dithiothreitol for 60 min at 40 [degrees]C. Samples were then alkylated with 50 mmol/L iodoacetamide for 60 min in the dark at room temperature. After reduction and alkylation we decreased the urea concentration by diluting the samples to 490 [micro]L with 50 mmol/L hydroxymethylaminoethane, pH 8, containing 10 mmol/L CaCl2. We then added 1 mg of L-(tosylamido-2phenyl) ethyl chloromethyl ketone-treated trypsin (Sigma-Aldrich) and digested the sample overnight at 37 [degrees]C.

LC-MS/MS

A Waters Q-TOF Premier quadrupole time-of-flight mass spectrometer was used to identify and acquire the relative abundances of all the tryptic peptides generated from the digest of recombinant ZAG. A more thorough explanation of the LC-MS/MS conditions used can be found elsewhere (15). The tryptic peptide from ZAG found to have the greatest response by this method was [sup.147][EIPAWVPFDPAAQITK.sup.162] (hereafter referred to as tpZAG147-162). The sequence EIPAWVPFDPAAQITK was found to be specific to Chain D, Human Zinc-A-2-Glycoprotein (accession no. 1ZAG D, gi:7246026) via a BLAST search, suggesting that this sequence could be used to specifically quantify ZAG. This same tryptic peptide for ZAG was recently described by Anderson and Hunter (16).

LC-MS/MS CONDITIONS

Absolute quantification was performed using a CTC Analytics HTC PAL autosampler (LEAP Technologies), a Shimadzu 10-AD binary pumping system, and an API 5000 triple quadrupole mass spectrometer (Applied Biosystems). For each run we injected a total of 10 [micro]L of sample onto a 50 x 2.1 mm TARGA [C.sub.18] column (Higgins Analytical) at a flow rate of 250 [micro]L/min, with a total run time of 30 min. The gradient used consisted of solvent A (water, 4 mmol/L ammonium acetate, 1 g/L formic acid) and solvent B (methanol, 4 mmol/L ammonium acetate, 1 g/L formic acid) starting at 5% B for 2 minutes, ramping to 95% B over 24 min, holding at 95% B for 2 min, back to 5% B in 1 min, and then holding to 30 min. The API 5000 instrument source parameters were CAD: 10, CUR: 30, GS1: 40, GS2: 40, IS: 5500, TEM: 600, DP: 225, and EP: 5. Using a 200 ms dwell time the doubly charged precursor ion for the ZAG tryptic peptide tpZAG147-162 at m/z 892 was selected in Q1 and 3 singly charged transitions were monitored in Q3: the y-ion PFDPAAQITK at m/z 1088 (CE: 40, CXP: 40), the y-ion PAAQITK at m/z 728.4 (CE: 40, CXP: 40), and the internal fragment ion PAW at m/z 355.2 (CE: 50, CXP: 26).

[FIGURE 1 OMITTED]

STABLE ISOTOPE-LABELED PEPTIDE INTERNAL STANDARD

The stable isotope peptide [sup.147][EIPAWVPFDPAAQITK.sup.162] was synthesized in the Mayo Proteomics Research Center on an ACT 396 Multiple Peptide Synthesizer (Advanced ChemTech), using recommended procedures for 1,3-diisopropylcarbodiimide activation and coupling. Stable isotope-labeled proline (5 [sup.13]C, 1 [sup.15]N-Fmoc-Proline, Isotech) was coupled in the peptide sequence at positions 7 and 9 to give a total molecular mass shift of +12 Da from the nonlabeled peptide and a monoisotopic molecular mass of 1775.92 Da. We added 2 [micro]L of the internal standard in concentration of 2 nmol/mL (3.6 [micro]g/mL) to 10 [micro]L of sample before injection into the LC-MS/MS.

LOCATION OF SELECTED PEPTIDE IN 3-DIMENSIONAL STRUCTURE OF ZAG

To coordinate the MS/MS measurements with potential immunoassay measurement systems, we specifically looked for a peptide sequence that was on the exterior of the 3-dimensional structure of ZAG. This exterior location would more likely be an immunologic binding site for appropriately targeted antisera, rather than an internal site. The IT7V Zn-[alpha]-2-glycoprotein:baculo-ZAG PEG 200 structure was downloaded from the RCSB Protein Data Bank (1). The structure was determined using x-ray diffraction with a 1.95 [Angstrom] resolution. The DeepView/ Swiss-PdbViewer 3.7 program (2) was used to visualize and highlight the epitope site [sup.147][EIPAWVPFDPAAQITK.sup.162] (17,18)

STUDY SUBJECTS

We used serum samples from 3 pilot groups of men in this study. The 1st group included healthy men (n = 6, ages 46 to 58 years), the 2nd group, men with nonmalignant prostate biopsy results (n = 20, ages 59 to 83 years), and the 3rd group, men with PCa (n = 26, ages 56 to 84 years). The men with nonmalignant prostate biopsy results had pathology reports of normal (n = 8), inflammation (n = 4), prostate intraepithelial neoplasia (n = 6), and atypical acinar proliferation (n = 2). Median follow-up of the 29 men with PCa was 26.5 months (range 3.8-43.6 months). The prostate-specific antigen values were [less than or equal to]4 [micro]g/L (2 patients); 4.1-10 [micro]g/L (6 patients); 10.1-20 [micro]g/L (7 patients); and >20 [micro]g/L (11 patients). Gleason scores were available for 25 patients at the time of initial diagnosis; 36% had a total Gleason score of 6, 32% a score of 7, 16% a score of 8, and 16% a score of 9 or 10. We obtained the specimens from the Mayo Foundation Prostate Specialized Program in Research Excellence (SPORE). These specimens were collected in 2002-2006, according to a protocol approved by Mayo Clinic Institutional Review Board (#1937-00).

CALCULATIONS AND STATISTICS

We defined the limit of quantification (LOQ) for ZAG as the response for digested recombinant ZAG added to 50 g/L BSA matrix that gave a signal-to-noise value of 10. The limit of detection (LOD) for ZAG was defined as the concentration of standard that gave a signal-to-noise value of 3. We used 2-tailed Student t-tests (SAS software) to compare the ZAG concentrations in the 3 pilot study groups.

Results

The most critical step in the protein cleavage approach to quantification of a protein is the selection of a cleaved peptide that will provide adequate analytical specificity and sensitivity. Selection of the tryptic peptide tpZAG147-162 for ZAG quantification was determined empirically by performing LC-MS/MS on tryptic digests of recombinant ZAG. The most abundant tryptic peptide observed was tpZAG147-162 (Fig. 1A). Fortuitously, this tryptic peptide has 3 proline residues that produce strong fragment ions with m/z values of 355.2, 728.5, and 1088.7 Da (Fig. 1B). All ZAG quantification was performed using the [y.sub.10] = 1088.7 fragment ion response since this transition had the best signal-to-noise and LOQ for ZAG from human serum. Also fortuitous was the location of this peptide on the external part of the molecule (see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue4).

Quantification was performed using a standard curve prepared by serial dilutions of recombinant ZAG added to 50 g/L BSA in phosphate-buffered saline as a surrogate serum matrix. Fig. 2 shows 3 ion chromatograms: (a) the digest of recombinant ZAG added to 50 g/L of BSA at a concentration of 1.3 mg/L, (b) ZAG tryptic peptide produced after the digest of patient serum, and (c) the stable isotope-labeled tpZAG147-162 added to the ZAG standard after digestion. We added 2 [micro]L of the internal standard in concentration of 2 [micro]mol/L (3.6 mg/L) to 10 [micro]L of sample. A comparison of the response for equimolar amounts of ZAG vs the internal standard peptide was performed by generating a standard curve, with each point in the curve having equimolar amounts of ZAG and internal standard. Linear regression analysis of this curve resulted in y = 1.054x - 0.283 with an [R.sup.2] = 0.9965. These results demonstrated that ZAG was completely digested in the 50 g/L BSA matrix, as the regression line slope was ~1.0 and the intercept reflected minimal background interference. After standard curves were found to be linear and reproducible, patient and normal donor serum samples were digested and analyzed.

[FIGURE 2 OMITTED]

The trace for the PCa patient (Fig. 2B) shows the superior signal-to-noise afforded by the [y.sub.10] transition for the tpZAG147-162 peptide. Controls run without supplementing ZAG or stable isotope-labeled tpZAG147-162 into 50 g/L BSA matrix showed low background response for the [y.sub.10] transition, as did controls for the internal standard transition in human serum digests where no internal standard was added. Since ZAG free human serum was not available, standard addition of recombinant ZAG into pooled normal male sera was performed to further confirm the origin of the peak observed in serum digests. Recoveries were also determined by adding recombinant ZAG to pooled normal male sera. Two different supplemented concentrations were evaluated, 3 mg/L (within normal range) and 6 mg/L (mean value for nonmalignant prostate disease). The results are shown in Table 1. In addition, a 5-point dilution series was made from a nonmalignant prostate serum sample by adding 50 g/L BSA matrix. Linear regression analysis of the dilution series, plotted as the known concentration vs the calculated concentration, was found to be y = 0.8112x + 2.063 with an [R.sup.2] = 0.9695. Fig. 3 shows a typical standard curve used for quantification, demonstrating the linearity associated with recombinant ZAG added to the BSA matrix over a range of 0.33 to 10.4 mg/L. The LOQ was 0.32 mg/L and the LOD was 0.08 mg/L. This LOD translates into ~20 fmol of tpZAG147-162 loaded on column for a 10 [micro]L injection.

To evaluate intra- and interassay imprecision, we prepared 2 serum pools from normal male and normal female sera. Aliquots of these pools were reduced, alkylated, and digested separately and then analyzed using our LC-MS/MS method over a period of 3 consecutive days. The results of this analysis (Table 1) demonstrated the reproducibility of our method; 10 separate digests performed and run over 3 consecutive days showed CVs <7%. The ZAG concentration from normal male sera was between 2.6 and 4.7 mg/L with an average concentration of 3.65 mg/L. The average concentration of ZAG in men with nonmalignant prostate disease was 6.21 mg/L, and the average ZAG concentration in men with PCa was 7.59 mg/L. Student t-tests performed on the 3 sample sets comparing samples of normal, nonmalignant disease, and PCa samples showed statistically significant increases in the concentration of ZAG across these groups (Table 2) when analyzed by our LC-MS/MS method.

Discussion

The method presented here for the quantification of ZAG in serum by LC-MS/MS is based on the technique first described by Barr et al. (19) and modified by our group and others for use in quantifying proteins from serum (16,20). Recently, Anderson and Hunter described the same tryptic fragment of ZAG protein (tpZAG147-162) that can be used for identification of ZAG among 53 plasma proteins ranging in abundance from albumin (~70 g/L) down to fibronectin (~1 mg/L) (16). We performed absolute quantification of ZAG by using pure recombinant protein for our standards, and a stable isotope-labeled synthetic peptide with the same sequence as tryptic fragment tpZAG147-162 as an internal standard. The concentration of ZAG in serum is relatively high (our findings showed ~3-8 mg/L), making it possible to quantify the protein by LC-MS/MS directly from digests of serum, without purification or depletion of high-abundance proteins. We were also able to use a high LC flow rate of 250 [micro]L/min and an electrospray source, both of which are routinely used in clinical laboratories. We anticipate that the method will be a valuable resource for determining the concentration of ZAG in larger cohorts of patients to determine the utility of ZAG as a cancer biomarker.

[FIGURE 3 OMITTED]

If follow-up LC-MS/MS cohort studies demonstrate the ZAG protein has clinical utility as a cancer biomarker, development of an immunoassay may be desirable. The position of the tpZAG147-162 fragment on the periphery of the proteins tertiary structure suggests that this peptide may also provide an accessible epitope for antibody binding and facilitate future assay development. This LC-MS/MS assay, based on an external peptide, could potentially be used as a reference method for immunoassays, especially if the immunoassays also target the same external region of the ZAG molecule illustrated in Fig. 1 in the online Data Supplement.

ZAG has been identified as a biomarker associated with multiple diseases, including prostate, bladder, and breast cancer (3, 6, 7). Therefore, ZAG concentrations by themselves are unlikely to be a specific screening test for any targeted disease, such as PCa. Nonetheless, ZAG may be a valuable biomarker to increase sensitivity for early disease detection if it is combined with other more tissue specific biomarker markers, such as prostate-specific antigen. ZAG also may have utility for differentiating more aggressive forms of cancer. However, a large number of measurements will be needed to further define the screening or prognostic value of ZAG, and the LC-MS/MS assay described in this paper should be a valuable tool for conducting these studies.

This study was partially supported by SPORE in Prostate Cancer Grant P50CA 91956 from the National Cancer Institute, NIH.

Received September 7, 2006; accepted January 18, 2007. Previously published online at DOI: 10.1373/clinchem.2006.079681

References

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(2.) Sanchez LM, Chirino AJ, Bjorkman P. Crystal structure of human ZAG, a fat-depleting factor related to MHC molecules. Science 1999;283:1914-9.

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(4.) Russell ST, Tisdale MJ. The role of glucocorticoids in the induction of zinc-alpha2-glycoprotein expression in adipose tissue in cancer cachexia. Br J Cancer 2005;92:876-81.

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(8.) Sanchez LM, Vizoso F, Diez-Itza I, Lopez-Otin C. Identification of the major protein components in breast secretions from women with benign and malignant breast diseases. Cancer Res 1992;52: 95-100.

(9.) Davidsson P, Nilsson CL. Peptide mapping of proteins in cerebrospinal fluid utilizing a rapid preparative two-dimensional electrophoretic procedure and matrix-assisted laser desorption/ionization mass spectrometry. Biochim Biophys Acta 1999;1473:391-9.

(10.) Hirai K, Hussey HJ, Barber MD, Price SA, Tisdale MJ. Biological evaluation of a lipid-mobilizing factor isolated from the urine of cancer patients. Cancer Res 1998;58:2359-65.

(11.) Tada T, Ohkubo I, Niwa M, Sasaki M, Tateyama H, Eimoto T. Immunohistochemical localization of Zn-alpha 2-glycoprotein in normal human tissues. J Histochem Cytochem 1991;39:1221-6.

(12.) Hale LP, Price DT, Sanchez LM, Demark-Wahnefried W, Madden JF. Zinc alpha-2-glycoprotein is expressed by malignant prostatic epithelium and may serve as a potential serum marker for prostate cancer. Clin Cancer Res 2001;7:846-53.

(13.) Irmak S, Tilki D, Heukeshoven J, Oliveira-Ferrer L, Friedrich M, Huland H, et al. Stage-dependent increase of orosomucoid and zinc-alpha2-glycoprotein in urinary bladder cancer. Proteomics 2005;5:4296-304.

(14.) Ekman R, Johansson BG, Ravnskov U. Renal handling of Znalpha2-glycoprotein as compared with that of albumin and the retinol-binding protein. J Clin Invest 1976;57:945-54.

(15.) Barnidge DR, Tschumper RC, Jelinek DF, Muddiman DC, Kay NE. Protein expression profiling of CLL B cells using replicate off-line strong cation exchange chromatography and LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 2005;819:33-9.

(16.) Anderson L, Hunter CL. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics 2006;5:573-88.

(17.) Guex N, Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 1997;18:2714-23.

(18.) Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The Protein Data Bank. Nucleic Acids Res 2000;28:235-42.

(19.) Barr JR, Maggio VL, Patterson DG Jr, Cooper GR, Henderson L0, Turner WE, et al. Isotope dilution-mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I. Clin Chem 1996;42:1676-82.

(20.) Barnidge DR, Goodmanson MK, Klee GG, Muddiman DC. Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-Ms/MS using protein cleavage and isotope dilution mass spectrometry. J Proteome Res 2004;3:644-52.

[1] Nonstandard abbreviations: ZAG, zinc-a2 glycoprotein; LC-MS/MS, liquid chromatography-tandem mass spectrometry; PCa, prostate cancer; BSA, bovine serum albumin; LOQ, limit of quantification; LOD, limit of detection.

OLGA P. BONDAR, DAVID R. BARNIDGE, ERIC W. KLEE, BRIAN J. DAVIS, AND GEORGE G. KLEE *

Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, MN.

* Address correspondence to this author at: Mayo Clinic and Mayo Foundation, 200 First St. SW, Rochester, MN 55905. Fax 507-284-4542; e-mail klee.george@mayo.edu.
Table 1. Assay performance characteristics.

Recovery in normal male pool

Added, [micro]g Measured, mg/L % Recovery
0 2.98
3 5.76 93%
6 9.8 114%

 Intra- and interassay precision

 Intraassay

 Male pool Female pool
Replicates 5 5
Mean, mg/L 3.27 3.42
SD, mg/L 0.16 0.22
CV, % 4.99 6.30

 Intra- and interassay precision

 Interassay

 Male pool Female pool
Replicates 15 15
Mean, mg/L 3.26 3.38
SD, mg/L 0.14 0.20
CV, % 4.36 5.92

Table 2. Comparison of serum ZAG values in normal
individuals, patients with nonmalignant prostate disease,
and patients with PCa.

 Serum ZAG concentration,
 mg/L

Group Number Average SD P values
Normal 6 3.65 0.71 0.0013

Nonmalignant prostate 20 6.21 1.65
disease 0.037
 0.0007
PCa 25 7.59 2.45
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Title Annotation:Cancer Diagnostics
Author:Bondar, Olga P.; Barnidge, David R.; Klee, Eric W.; Davis, Brian J.; Klee, George G.
Publication:Clinical Chemistry
Date:Apr 1, 2007
Words:3387
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