Quantitative Analysis of an Aberrant Glycoform of TIMP1 from Colon

Jul 6, 2009 - systematic coupling of L-PHA lectin enrichment followed by stable isotope standards ..... 0. 1. 2 av. area ratio, log(L/H). -3.21 -2.50 ...
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Quantitative Analysis of an Aberrant Glycoform of TIMP1 from Colon Cancer Serum by L-PHA-Enrichment and SISCAPA with MRM Mass Spectrometry Yeong Hee Ahn,† Ji Yeon Lee,† Ju Yeon Lee,† Yong-Sam Kim,‡ Jeong Heon Ko,‡ and Jong Shin Yoo*,†,¶ Division of Mass Spectrometry, Korea Basic Science Institute, 804-1 Yangcheong-Ri, Ochang-Myun, Cheongwon-Gun 363-883, Republic of Korea, GRAST, Chungnam National University, Daejeon 305-764, Republic of Korea, and Daejeon-KRIBB-FHCRC Research Cooperation Center, KRIBB, 111 Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea Received February 18, 2009

Variations in glycosylation levels or in the glycoprofile of a certain glycoprotein in tumor-related sera have been widely reported and can be used as a means of differentiation. However, quantitative mass analysis of glycoproteins is difficult because of their high structural complexity and low mass sensitivity of glycopeptides. Therefore, more powerful technologies are required for the discovery of these potential biomarkers. Tissue inhibitor of metalloproteinase 1 (TIMP1), a glycoprotein typically present at a low concentration in serum, is known to be aberrantly glycosylated in colorectal cancer cell lines as a result of the terminal addition of β-1,6-N-acetylglucosamine (β-1,6-GlcNAc) by N-acetylglucosaminyltransferase-V (GnT-V), which is reportedly up-regulated in invasive/metastatic cancer cells. In this report, a highly sensitive method is presented for the quantitative analysis of aberrant GlcNAcylated TIMP1 in the serum of colorectal cancer (CRC) patients. Glycoproteins having an N-linked glycan terminating with β-1,6-GlcNAc were enriched by phytohemagglutinin-L4 (L-PHA), a lectin that specifically recognizes the β-1,6-GlcNAc moiety of N-linked glycan. The L-PHA-enriched glycoproteins were digested in solution with trypsin. With the use of a monoclonal anti-peptide TIMP1 antibody linked covalently to magnetic beads, a unique target peptide (antigen) of TIMP1 was immuno-enriched from the L-PHA-enriched tryptic digests and analyzed quantitatively by multiple reaction monitoring (MRM) mass analysis. The systematic coupling of L-PHA lectin enrichment followed by stable isotope standards and capture by anti-peptide antibodies (SISCAPA) with MRM mass analysis afforded quantitation of TIMP1 at attomolar (10-18) concentrations. An aberrantly GlcNAcylated substoichiometric TIMP1 isoform was quantified at approximately 0.8 ng/mL serum, using sample equivalent to only 1.7 µL of serum from a CRC patient. This approach provides a useful tool for the quantitation of a specific aberrant glycoform from human serum containing a variety of protein isoforms and may be helpful in studies of biological function as it pertains to protein glycan heterogeneity. Keywords: peptide quantitation • aberrant glycoform • TIMP1 • L-PHA • anti-peptide antibody • SISCAPA

Introduction Protein glycosylation is one of the most important posttranslational modifications of proteins, and recent proteomic studies have shown that dynamic alterations to protein glycosylations are closely associated with pathogenic processes in cells.1-3 In some cases, aberrant glycosylation of a protein, which may include increased sialylation and fucosylation, results in increased size and branching of N-glycans in cancer * To whom correspondence should be addressed. Jong Shin Yoo, Division of Mass Spectrometry, Korea Basic Science Institute, 804-1 Yangcheong-Ri, Ochang-Myun, Cheongwon-Gun 363-883, Republic of Korea. Phone: +8243-240-5150. Fax: +82-43-240-5159. E-mail: [email protected]. † Korea Basic Science Institute. ‡ Daejeon-KRIBB-FHCRC Research Cooperation Center. ¶ GRAST, Chungnam National University.

4216 Journal of Proteome Research 2009, 8, 4216–4224 Published on Web 07/06/2009

cells.4-6 Cancer-induced aberrant glycosylation can be used as a biomarker for cancer.7 Although the use of biomarkers based on aberrant glycosylation represents a more accurate and sophisticated technique relative to changes in a specific protein abundance, the available methods with the required sensitivity for glycosylation analysis are limited, and the development of additional analytical tools is needed. N-acetylglucosaminyltransferase-V (GnT-V), a well-characterized glycosyltransferase associated with pathogenic processes in cells, is up-regulated in cancer cells and catalyzes the abnormal formation of a β-1,6-N-acetylglucosamine (β-1,6GlcNAc) moiety in an N-linked core glycan.4,8,9 An increase in the aberrant glycosylation induced by GnT-V is correlated with invasive/metastatic potentials in cancer cells. Tissue inhibitor of metalloproteinase 1 (TIMP1), a target molecule for GnT-V, 10.1021/pr900269s CCC: $40.75

 2009 American Chemical Society

Quantitation of an Aberrant Glycoform of TIMP1 is abnormally GlcNAcylated in cancer cells and may be involved in cancer progression.10 Despite its relationship with biological and pathological processes in cancers, TIMP1 has not yet been used as a cancer biomarker owing to the absence of technology which enables the distinction between the glycosylation profiles of normal and cancer sera, or to monitor trace changes in minute expression levels of the protein in blood. Thus, a more sensitive and selective technology is required to monitor changes in aberrant glycosylation in complex materials such as blood. Glycoproteins, which are aberrantly GlcNAcylated by GnTV, have an additional GlcNAc branch in their N-linked core glycan and can be enriched using phytohemagglutinin-L4 (LPHA), a lectin that specifically recognizes the β-1,6-GlcNAc moiety of N-linked glycan.11,12 Kim et al.13 applied the L-PHA lectin enrichment method in analyses of immuno-depleted sera of colorectal cancer (CRC) patients and reported that L-PHAenriched glycoproteins were candidate biomarkers for CRC. However, TIMP1, a target protein for GnT-V, was not identified in the mass analysis using an LC/FTLTQ mass spectrometer, which may be due to its low amount. Thus, in addition to the reduction in matrix complexity by the L-PHA lectin enrichment, further enrichment or extensive separation of the sample prior to analysis is required for mass identification of low-abundance serum proteins. Although a variety of immunoaffinity chromatographic technologies exist, the stable isotope standards and capture by anti-peptide antibodies (SISCAPA) method is particularly attractive for the specific enrichment of a target peptide for quantification by mass analysis.14 In contrast to protein-based techniques, this peptide-based immunoenrichment allows the qualitative and quantitative differentiation of heterogeneous protein isoforms and the characterization of differences due to post-translational modifications. With the SISCAPA approach, the immunoenrichment process can be combined with peptide-specific quantitation (MRM) methods by mass spectrometry,15-17 enabling the detection and quantitation of lowabundance proteins present within the nanogram per milliliter (ng/mL) range in serum.18 Enrichment by anti-peptide antibodies dramatically reduces the sample complexity of tryptic digests, allowing the target peptide to be analyzed with an increase in mass signal intensity of up to 3 orders of magnitude. It should be noted that, although the SISCAPA approach can be a powerful tool for the quantification of target peptides from a glycoprotein of interest, it does not provide information on changes in the glycoprofile of glycoproteins as discussed above for TIMP1. The current study describes a technique for the quantitation of low-abundance TIMP1 isoforms with aberrant GlcNAcylated glycan moieties from CRC serum samples. The technique combines L-PHA-based enrichment of aberrantly glycosylated proteins and SISCAPA of the tryptic digests of the enriched glycoproteins, followed by mass analysis (Figure 1).

Experimental Section Materials. All reagents, including R-casein (bovine), β-casein (bovine), and ovalbumin (chick), were purchased from SigmaAldrich unless otherwise noted. Monoclonal antibodies against the tryptic peptides GFQALGDAADIR and SEEFLIAGK of human TIMP1 were manufactured by ABFrontier (Seoul, Korea) following the process of Supplement 1 in Supporting Information. The stable isotope-coded peptides GFQALGDAADIR and SEEFLIAGK (isotopically labeled sites in italics, 13C and 15N incorporated) were obtained from Anygen Co. (Kwangju,

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Figure 1. This block diagram shows the workflow for the quantitation of aberrantly glycosylated TIMP1, using lectinmediated enrichment and a SISCAPA approach with MRM mass analyses.

Korea). The isotope-labeled peptides are detected by a +4 Da shift corresponding to the singly charged peptides in the mass spectra. Dynabead MyOne tosylactivated magnetic beads with preactivated and tosylated surfaces were purchased from Invitrogen. Trypsin (sequencing grade) was purchased from Promega. Trypsin inhibitor, N-R-Tosyl-Lys chloromethyl ketone (TLCK), was obtained from Calbiochem. Immobilization of Anti-Peptide Antibody on Magnetic Beads. Anti-peptide TIMP1 antibody was immobilized on the tosylactivated magnetic beads according to the manufacturer’s instructions. Briefly, the antibody was added to a suspension of magnetic beads (40 µg antibody/mg bead) that had been prewashed with a coating buffer (0.1 M sodium borate, pH 9.5), and the beads were agitated at room temperature for 5 min. After the addition of 3 M ammonium sulfate buffer, the reaction tube was incubated at 37 °C for 16 h with slow agitation, to allow the antibody to covalently bind to the bead surface. The bead supernatant was removed, and the beads were resuspended in a blocking buffer (PBS, pH 7.4 with 0.5% (w/v) BSA and 0.05% Tween 20), followed by further incubation at 37 °C for 16 h with slow agitation. The bead-bound antibody was washed thoroughly with PBS (× 3) and stored at 4 °C in buffer (PBS, pH 7.4 with 0.1% (w/v) BSA and 0.02% (w/v) sodium azide). Capturing Efficiency of Immobilized Antibody. The enrichment activity of the immobilized antibody was validated with the synthetic peptide GFQALGDAADIR. Standard proteins (each 200 µg) were trypsin-digested separately (protein/trypsin ) 40: 1, w/w) in 50 mM ammonium bicarbonate buffer and combined to dry in vacuum. The tryptic digests was reconstituted to 6.3 µg/µL with 50 mM ammonium bicarbonate. To each of six microtubes containing a 60 µg sample/tube, TLCK (1 mM, 2.4 µL) was added, and the tubes were incubated at room temperature for 6 h to allow the trypsin to be inactivated. Increasing amounts (0.04, 0.4, 4, 40, 400, and 4000 fmol) of the synthetic target peptide (light) were added to each tube. The tryptic digests from each tube were mixed with bead-immobilized antibody (500 µg beads) that had been washed in advance with PBS (× 3). After incubation at room temperature for 1 h with slow agitation, the bead supernatant was removed. The beads were thoroughly washed with PBS (× 3), and the peptide that had bound to the immobilized antibody was eluted with an elution buffer (0.1 M glycine-HCl, pH 1.5). The stable Journal of Proteome Research • Vol. 8, No. 9, 2009 4217

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isotope-coded internal standard peptide (heavy, 40 fmol) was then added to each eluted solution, and one-fourth of each solution was analyzed using a LC/ESI-FTLTQ mass spectrometer. The experiment was performed in triplicate. Preparation of CRC Serum Samples by Lectin Enrichment. Serum samples were prepared from colon cancer patients at Our Lady of Mercy Hospital at The Catholic University of Korea (Incheon), with an agreement of participation from all subjects. Serum was diluted 2- to 3-fold with filter-sterilized distilled water and subjected to partial purification using a ProteomPrep 20 plasma immunodepletion LC column kit (Sigma). The flowthrough fraction was reduced by treatment with 1% (v/v) β-mercaptoethanol and alkylated with 50 mM iodoacetamide at room temperature for 1 h. The modified samples were desalted on a HiPrep 26/10 desalting column (GE Healthcare) and concentrated to a final volume of 1 mL. One milligram of depleted proteins measured by a BCA assay was precleared with avidin-agarose beads (Sigma) for 1 h at room temperature, and the precleared proteins were allowed to bind to L-PHA-avidinagarose overnight at 4 °C. L-PHA-avidin-agarose beads were prepared in advance by mixing biotinylated L-PHA (Sigma) with the avidin-agarose beads for 2 h at room temperature. After extensive washing with PBS, the bound proteins were separated from the bead complexes by adding 6 M urea. After a 10-fold dilution with distilled water, the protein preparation was digested with 20 µg of trypsin (depleted proteins/trypsin ) 50: 1, w/w) in 50 mM ammonium bicarbonate and 1 mM CaCl2 overnight at 37 °C. The digested peptides were lyophilized in a SpeedVac system before being analyzed by mass spectrometry. Antibody Enrichment of CRC Serum. Tryptic digests of the lectin-enriched CRC serum were reconstituted in 50 mM ammonium bicarbonate. TLCK was added to the partitioned tryptic digests, and the peptide solutions were incubated at room temperature for 6 h. After spiking with the internal standard peptide (heavy, 40 fmol), the tryptic digests were mixed with bead-immobilized antibody (500 µg beads) that had been prewashed with PBS (3 × 200 µL). After incubation at room temperature for 1 h with slow agitation, the supernatant was removed. The beads were washed thoroughly with PBS (3 × 200 µL), and the enriched peptides were eluted with an elution buffer (0.1 M glycine-HCl, pH 1.5). One-fourth of eluted solution was analyzed in triplicate, using a LC/ESI-FTLTQ mass spectrometer. Liquid Chromatography. The separation and analysis of the tryptic peptides that eluted from the bead-immobilized antibody were performed using a nano-LC/MS system consisting of a Surveyor HPLC (ThermoFinnigan) and a FTLTQ mass spectrometer (ThermoFinnigan) equipped with a nano-ESI source. Mobile phases A and B were composed of 0% and 80% acetonitrile, respectively, each containing 0.5% acetic acid and 0.02% formic acid. Gradient elution was performed as follows: 5% B for 15 min, 20% B for 3 min, 50% B for 47 min, 95% B for 7 min, and 5% B for 2 min. The column was equilibrated with 5% B for 8 min between each gradient LC run. A 10-µL aliquot of each peptide solution in mobile phase A was loaded onto a C18 trap column (pore size, 5 µm; 300 µm i.d. × 5 mm, LC Packing) by an autosampler, and the column was washed with mobile phase A for 10 min at 20 µL/min. The peptides were in-line transferred onto a homemade C18 column (Phenomenex Aqua, 5 µm; 75 µm i.d. × 100 mm; 6 µm orifice i.d.), eluted with the aforementioned gradient, and directly electrosprayed into the FTLTQ mass spectrometer, controlled by Xcalibur software (ThermoElectron). 4218

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Figure 2. The linear ranges of mass analyses are shown for mixture solutions of a synthetic peptide GFQALGDAADIR (light, L) and its stable isotope-coded (heavy, H). The mass analyses were performed in triplicate for each ratio of peptides injected.

Mass Analysis. The identification of proteins in tryptic digests that had been enriched by L-PHA but not by the beadimmobilized antibody was conducted in triplicate, using samples equivalent to 100 µg of the depleted protein. One full mass spectrum was acquired in the range of 400-2000 m/z. Three ion-trap MS/MS spectra were acquired per data-dependent cycle. The mass analysis method used was as follows: ion spray voltage 2.2 kV, capillary temperature 220 °C, 1 FTMS full micro scan with 200 ms FTMS full max ion time, 3 ion trap MSMS micro scans with 50 ms ion trap MSMS max ion time. The dynamic exclusion settings used for MSMS were as follows: repeat count 1, exclusion list size 25, exclusion duration 60 s, exclusion mass width (by mass) low 2 and high 2. The collisioninduced dissociation (CID) MSMS were conducted with 35% normalized collision energy, 3000 counts minimum signal threshold, 2 isolation width, 0.25 activation Q, and 30 ms activation time for MSMS acquisitions. The.xml data file generated by Bioworks software (ThermoElectron) was submitted to the Mascot search engine (version 2.1, Matrix Science), using the Swiss-Prot 51.6 database confined to Homo sapiens taxonomy. The Mascot search was performed with monoisotopic mass, a precursor mass tolerance of 0.2 Da, and a fragment mass tolerance of 0.4 Da. Trypsin was selected as the enzyme, with consideration of two potentially missed cleavages and a possible cleavage at the K/R-P amide bond. Carbamidomethylated cysteine was selected as a fixed modification. Oxidized methionine and pyro-glutamate (N-term Q) were selected as variable modifications. A Mascot score of 33 (p < 0.05) was used as the acceptance criterion for peptide identification. For quantitative MRM analyses, MS/MS spectra were first obtained for the synthetic target peptide GFQALGDAADIR (light) and its stable isotope-coded counterpart (heavy). And three pairs of transitions showing strong signal intensities with isotopically differentiated residue were selected from among the many fragment ion signals in each MS/MS spectrum for the MRM mass assay. For the target peptide GFQALGDAADIR (2+, m/z 617.31), the peaks at m/z 660.36, 717.35, and 831.48 were selected. For the stable isotope-coded standard peptide GFQALGDAADIR (2+, m/z 619.31, 13C and 15N

Quantitation of an Aberrant Glycoform of TIMP1

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Figure 3. The MS/MS spectra show the fragmentation patterns of (a) the synthetic target peptide, GFQALGDAADIR (light), and (b) its stable isotope-coded analogue (heavy). The two peptides show essentially the same fragmentation pattern, except for a 4 Da mass shift due to the isotope-coded alanine residue (in italics). Three transitions were selected for MRM mass analysis. For the target peptide (light, m/z 617.31), the peaks at m/z 660.36, 717.35, and 831.48 were selected (*marked on fragment ions). For the stable isotope-coded (heavy, m/z 619.31) analogue, the peaks at m/z 664.45, 721.38, and 834.50 were selected (*marked on fragment ions).

incorporated in the alanine residue shown in italics), the peaks at m/z 664.45, 721.38, and 834.50 were selected. The inclusion window was (1 Da for each precursor ion, and (2 Da for the fragment ions. MRM analyses were conducted by examining the chromatographic peak area ratios of the selected transition pairs. Among the three pairs of selected channels, the most abundant transition was used for quantitation.

Results and Discussion Preparation of Target Peptides. TIMP1 is a glycoprotein with two N-glycan moieties, at Asn30 and Asn78, and multiple

disulfide linkages. It was identified based on a few tryptic peptides by ESI mass analysis in an earlier paper.10 Since Two tryptic peptides, having the sequences GFQALGDAADIR and SEEFLIAGK, are consistently identified with relatively strong signal intensities and considered not to contain any residue of post-translational modification, these peptide was selected as the antigens for the preparation of anti-peptide antibodies. With the use of the synthetic target peptide having the sequence GFQALGDAADIR and its stable isotope-coded counterpart, the relative content of prepared stark solution of each peptides and the linearity of quantitative mass analysis was Journal of Proteome Research • Vol. 8, No. 9, 2009 4219

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Figure 4. Blank test in MRM mass analysis using a sample containing 10 fmol of the internal standard (heavy) but no light peptide. The X-axes of the displayed chromatograms are expanded to the elution time of the internal standard. (a) Represents chromatograms obtained for the transitions, m/z 617.31 f 717.35 for light peptide and m/z 619.31 f 721.38 for heavy peptide. In the monitoring channel for the transition of the light peptide, no signal due to interference from the spiked internal standard (heavy) was observed. (b) Represents spectra of selected fragment ions of the heavy peptide. The transition, m/z 619.31 f 721.38, shows the most intensity.

Table 1. Quantitation by the SISCAPA Mass Analysis of the Target Peptide Spiked into Tryptic Digests of Three Standard Proteins

Figure 5. The detection limit and linear concentration range as determined from MRM mass analysis of the antigen GFQALGDAADIR spiked to tryptic digests of three standard proteins are shown. The curve is displayed using the chromatographic peak area ratios of the transition pairs, the m/z 617.31 f 717.35 transition of the endogenous antigen (light) and the m/z 619.31 f 721.38 transition of the internal standard (heavy). 4220

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antigen, L standard, H peptide ratio, log(L/H)

0.01 10 -3

0.1 10 -2

1 10 -1

10 10 0

100 10 1

1000 10 2

av. area ratio, log(L/H) SD, %

-3.21

-2.50

-1.41

-0.51

0.36

1.42

2.5

3.3

4.2

3.8

3.0

2.4

examined. The response of the mass analysis showed good linearity (R2 ) 0.9987) over 4 orders of magnitude, from 1 pmol to 0.1 fmol, of peptide (Figure 2). Sensitivity and Linear Range of Immobilized Antibody Analyses. The detection limit and linear concentration range of antigen mass analyses can be influenced by several factors such as the antigen-capturing efficiency of the antibody, the chromatographic separation efficiency, and the mass sensitivity and dynamic range of the mass spectrometer. The capturing efficiency of the anti-peptide TIMP1 antibody against the synthetic antigen GFQALGDAADIR was determined from MRM

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Quantitation of an Aberrant Glycoform of TIMP1

Table 2. Identified Proteins Obtained from Full MS/MS Analyses of Tryptic Digests of L-PHA-Enriched CRC Serum no.

Swiss-Prot ID

protein name

Mascot score range

no. of ID

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

AACT_HUMAN APOA1_HUMAN HEMO_HUMAN ITIH2_HUMAN CFAB_HUMAN ITIH1_HUMAN VTNC_HUMAN IC1_HUMAN ANGT_HUMAN ANT3_HUMAN A2MG_HUMAN ITIH4_HUMAN VWF_HUMAN FINC_HUMAN GELS_HUMAN VTDB_HUMAN A1BG_HUMAN CFAH_HUMAN AFAM_HUMAN C1S_HUMAN K2C1_HUMAN LUM_HUMAN AMBP_HUMAN PGRP2_HUMAN A2GL_HUMAN CO9_HUMAN FETUA_HUMAN CLUS_HUMAN KNG1_HUMAN CO5_HUMAN CO4A_HUMAN CFAI_HUMAN APOE_HUMAN ITIH3_HUMAN PEDF_HUMAN HEP2_HUMAN CO6_HUMAN APOA4_HUMAN LG3BP_HUMAN K22E_HUMAN HRG_HUMAN THRB_HUMAN CO2_HUMAN K1C10_HUMAN K1C9_HUMAN A2AP_HUMAN KLKB1_HUMAN CBG_HUMAN APOH_HUMAN SAMP_HUMAN

2162-3051 1355-1849 1330-1570 622-1399 669-1125 565-1121 712-1025 708-840 575-736 473-665 36-615 381-578 244-552 299-548 351-535 424-522 378-453 384-457 208-444 344-436 287-422 220-379 229-366 117-362 164-350 264-325 156-325 295-324 209-311 183-303 109-297 103-279 76-275 179-266 164-256 155-246 69-236 112-235 90-225 135-220 134-203 125-194 11-184 89-179 125-173 60-173 62-164 75-163 94-159 103-158

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

THBG_HUMAN ALS_HUMAN CO8A_HUMAN CO7_HUMAN SAA4_HUMAN ALBU_HUMAN K2C5_HUMAN CPN2_HUMAN RETBP_HUMAN C4BP_HUMAN APOA2_HUMAN APOF_HUMAN FA10_HUMAN PROS_HUMAN MUCB_HUMAN

Alpha-1-antichymotrypsin precursor (ACT) Apolipoprotein A-I precursor (Apo-AI) Hemopexin precursor (Beta-1B-glycoprotein) Inter-alpha-trypsin inhibitor heavy chain H2 precursor Complement factor B precursor (C3/C5 convertase) Inter-alpha-trypsin inhibitor heavy chain H1 precursor Vitronectin precursor (Serum-spreading factor) Plasma protease C1 inhibitor precursor (C1 Inh) Angiotensinogen precursor Antithrombin-III precursor (ATIII) Alpha-2-macroglobulin precursor (Alpha-2-M) Inter-alpha-trypsin inhibitor heavy chain H4 precursor von Willebrand factor precursor (vWF) Fibronectin precursor (FN) (Cold-insoluble globulin) Gelsolin precursor (Actin-depolymerizing factor) Vitamin D-binding protein precursor (DBP) Alpha-1B-glycoprotein precursor (Alpha-1-B glycoprotein) Complement factor H precursor (H factor 1) Afamin precursor (Alpha-albumin) (Alpha-Alb) Complement C1s subcomponent precursor (C1 esterase) Keratin, type II cytoskeletal 1 (Cytokeratin-1) Lumican precursor (Keratan sulfate proteoglycan lumican) AMBP protein precursor N-acetylmuramoyl-L-alanine amidase precursor Leucine-rich alpha-2-glycoprotein precursor (LRG) Complement component C9 precursor Alpha-2-HS-glycoprotein precursor (Fetuin-A) Clusterin precursor (Complement-associated protein SP-40, 40) Kininogen-1 precursor (Alpha-2-thiol proteinase inhibitor) Complement C5 precursor Complement C4-A precursor (Acidic complement C4) Complement factor I precursor (C3B/C4B inactivator) Apolipoprotein E precursor (Apo-E) Inter-alpha-trypsin inhibitor heavy chain H3 precursor Pigment epithelium -derived factor precursor (PEDF) Heparin cofactor 2 precursor (Heparin cofactor II) Complement component C6 precursor Apolipoprotein A-IV precursor (Apo-AIV) Galectin-3-binding protein precursor Keratin, type II cytoskeletal 2 epidermal (Cytokeratin-2e) Histidine-rich glycoprotein precursor Prothrombin precursor (Coagulation factor II) Complement C2 precursor (C3/C5 convertase) Keratin, type I cytoskeletal 10 Keratin, type I cytoskeletal 9 Alpha-2-antiplasmin precursor (Alpha-2-plasmin inhibitor) Plasma kallikrein precursor (Kininogenin) Corticosteroid-binding globulin precursor (Transcortin) Beta -2-glycoprotein 1 precursor (Apolipoprotein H) Serum amyloid P-component precursor (9. 5S alpha -1glycoprotein) Thyroxine-binding globulin precursor (T4-binding globulin) Insulin-like growth factor-binding protein Complement component C8 alpha chain precursor Complement component C7 precursor Serum amyloid A-4 protein precursor (C-SAA) Serum albumin precursor Keratin, type II cytoskeletal 5 (Cytokeratin-5) Carboxypeptidase N subunit 2 precursor Plasma retinol-binding protein precursor (PRBP) C4b-binding protein alpha chain precursor Apolipoprotein A-II precursor (Apo-AII) Apolipoprotein F precursor (Apo-F) Coagulation factor X precursor (Stuart factor) Vitamin K-dependent protein S precursor Ig mu heavy chain disease protein (BOT)

84-136 73-123 52-146 34-127 77-111 77-106 86-98 72-98 51-98 35-98 47-96 50-94 56-86 58-71 47-65

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

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Table 2. Continued no.

Swiss-Prot ID

protein name

Mascot score range

no. of ID

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82

CBPN_HUMAN CHL1_HUMAN IBP3_HUMAN HABP2_HUMAN FHR3_HUMAN ATRN_HUMAN C1R_HUMAN APOC3_HUMAN FBLN1_HUMAN HGFA_HUMAN MUC_HUMAN KAIN_HUMAN BTD_HUMAN APOC1_HUMAN CO8G_HUMAN CBPB2_HUMAN PHLD1_HUMAN

60-62 40-61 55-60 48-50 36-46 38-45 111-147 55-111 98-105 68-96 88-95 88-92 83-90 44-88 46-66 55-61 48-60

3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2

83 84 85 86 87 88 89

FHR2_HUMAN FCN3_HUMAN HORN_HUMAN FHR1_HUMAN THYG_HUMAN SCYB7_HUMAN MBD3_HUMAN

Carboxypeptidase N catalytic chain precursor Neural cell adhesion molecule L1-like protein precursor Insulin-like growth factor-binding protein 3 precursor Hyaluronan-binding protein 2 precursor Complement factor H-related protein 3 precursor Attractin precursor (Mahogany homologue) Complement C1r subcomponent precursor Apolipoprotein C-III precursor (Apo-CIII) Fibulin-1 precursor Hepatocyte growth factor activator precursor (HGF activator) Ig mu chain C region Kallistatin precursor Biotinidase precursor Apolipoprotein C-I precursor (Apo-CI) (ApoC-I) Complement component C8 gamma chain precursor Carboxypeptidase B2 precursor (Carboxypeptidase U) Phosphatidylinositol-glycan-specific phospholipase D 1 precursor Complement factor H-related protein 2 precursor (FHR-2) Ficolin-3 precursor Hornerin Complement factor H-related protein 1 precursor Thyroglobulin precursor Platelet basic protein precursor (PBP) Methyl-CpG-binding domain protein 3

46-59 49-57 53 46-51 39-40 33-39 34-36

2 2 2 2 2 2 2

mass analyses of trypsin-digested mixtures of three standard proteins (R-casein, β-casein, and ovalbumin) spiked in advance with different amounts of antigen. Each antigen-spiked sample was enriched by mixing with an equal amount of beadimmobilized antibody. MRM samples were prepared by spiking an equal amount of the internal standard (heavy peptide, 40 fmol) to each solution eluted from the antibody, and one-fourth of each sample was injected to do mass analysis. The MS/MS spectra shown in Figure 3 were obtained from the synthetic target peptide GFQALGDAADIR (light) and its stable isotopecoded form (heavy). The two peptides exhibited essentially the same fragmentation pattern, except for a 4 Da mass shift for fragment ions containing the isotope-coded alanine residue. Three transitions, corresponding to the fragment ions that exhibited strong signal intensities in each spectrum, were selected for MRM mass analysis. First, blank experiment was conducted using a sample containing the internal standard (heavy peptide, 10 fmol) but no light peptide. From monitoring the most abundant transition, the m/z 617.31 f 717.35 among the selected MRM channels for the light peptide, it was confirmed that no signal due to the light peptide was recorded and no interference due to the internal standard (heavy) was observed in monitoring of the transition of the light peptide (Figure 4). Figure 5 shows a calibration curve obtained by MRM mass analysis of the prepared tryptic digests of the three standard proteins. Good linearity (R2 ) 0.9977) was observed over 5 orders of magnitude, from 1 pmol to 10 amol, of the spiked synthetic antigen, together with low relative errors (Table 1). This implies that, under the enrichment conditions employed, the capturing efficiency of the antibody was not influenced seriously by the absolute amount of spiked antigen as well as the ratio of spiked antigen to antibody. Although the mixture of three standard proteins does not accurately represent a complex mixture such as serum, average capturing efficiency of the SISCAPA is about 36% under the enrichment conditions employed. The amount of 10 amol of the spiked antigen 4222

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peptide corresponds to 12 fg (approximately equivalent to 300 fg of TIMP1). TIMP1 is present at low concentrations (50-80 ng/mL) in human blood but may be found at higher concentrations in cancer patients.19 Serum TIMP1 is secreted from a variety of organs, including liver, stomach, and colon, and exists as isoforms with diverse glycan heterogeneity. While the overall serum concentration of TIMP1 is normally very low, the amount of aberrantly glycosylated TIMP1 produced in cancer tissues of the secreting organs is most likely even lower. Therefore, high sensitivity is a critical factor for successful quantitation of the aberrantly glycosylated isoform. The sensitivity demonstrated in Figure 5 was determined to be sufficient for quantification of substoichiometric aberrant TIMP1 from a complex tryptic matrix of serum. A monoclonal antibody against the tryptic peptide SEEFLIAGK of human TIMP1 was also prepared, and its antigen-capturing efficiency was examined. Unfortunately, the antibody showed no activity against the antigen SEEFLIAGK, even though experiments were performed under a variety of conditions that served to enrich the antigen (data not shown). Quantitation of Aberrantly Glycosylated TIMP1 from CRC Serum. Glycoprotein TIMP1, a target protein for GnT-V in colon cancer cells, was found to be aberrantly glycosylated in a comparative study using two-dimensional electrophoresis and lectin blot analysis for secreted proteomes of the colon cancer cell line WiDr.10 An approach for high-throughput mass analysis of immuno-depleted and L-PHA-enriched CRC serum proteins was also introduced, but TIMP1 was not identified.13 In the current study, many proteins were identified in preliminary triplicate MS/MS analyses of immuno-depleted and L-PHA-enriched CRC serum samples, but TIMP1 was not detected. Proteins identified over duplicate experiments are listed in Table 2. This further demonstrates the difficulty in detecting the extremely low amount of the aberrantly GlcNAcylated TIMP1 isoform. In a separate experiment, triplicate MRM mass analyses were performed, without immunoenrichment by the anti-peptide

research articles

Quantitation of an Aberrant Glycoform of TIMP1

Table 3. Quantitation of the Target Antigen from Tryptic Digests of CRC Serum with L-PHA- and Immunoenrichmenta depleted proteins

10 µg injected

2.5 µg injected

run

amount, L (amol)

amount, L (amol)

1 2 3 average

45.8 47.4 43.8 45.7 (SD 3.9%)

10.3 8.6 7.7 8.9 (SD 14.6%)

estimated aberrant TIMP1

1.4 pg

0.3 pg

a The immuno-depleted proteins, 10 and 2.5 µg, were obtained by immunodepletion from serum corresponding to 1.67 and 0.42 µL, respectively. The mass of estimated TIMP1 were calculated using 30 kDa, Mw of TIMP1.

Figure 6. Filtered chromatograms were obtained from MRM mass analyses of tryptic digests of L-PHA-enriched CRC serum. The X-axes of the displayed chromatograms are expanded to the elution time of the internal standard. The chromatograms were filtered to display m/z 717.15-717.55 for the endogenous antigen (light, L) and m/z 721.15-721.55 for the spiked internal standard (heavy, H). Chromatograms (a) and (b) are representative of L-PHA-enriched samples from 100 µg and 50 µg of depleted sample, respectively, without immunoenrichment. Chromatogram (c) represents a sample subjected to both L-PHA- and immunoenrichment from 10 µg of depleted sample, and (d) shows the internal standard spike (H, 10 fmol).

TIMP1 antibody, with tryptic digests of L-PHA-enriched CRC serum samples equivalent to 100 or 50 µg of the immunodepleted protein. However, reliable quantification of the GFQALGDAADIR antigen was not possible (Figure 6a,b). The chromatogram peaks representing the m/z 617.31 f 717.35 transition of the endogenous antigen (light) were subject to severe interference from co-eluting fragment ions, regardless of the amount of sample injected. Therefore, efficient reduction of sample complexity is required for highly sensitive and reproducible MRM quantitation of the substoichiometric target peptide. At this time, the SISCAPA approach offers an alternative means of quantitative analysis for aberrantly GlcNAcylated TIMP1 after enrichment by L-PHA lectin. Immuno-depleted and lectin-enriched CRC serum was digested in-solution with trypsin. An equivalent amount of the standard peptide (heavy, 40 fmol) was spiked into each of the tryptic digests, which contained 40 and 10 µg of the immunodepleted proteins, respectively. After immunoenrichment by the anti-peptide TIMP1 antibody, triplicate MRM analyses were performed using one-fourth of each eluate solution. The results are summarized in Table 3 and Figure 6c,d. An aliquot of the depleted proteins (10 µg proteins) contained 45.7 amol of

GlcNAcylated TIMP1 protein (corresponding to approximately 1.4 pg of TIMP1) with good precision (standard deviation, 3.9%). Ten micrograms of proteins was obtained from about 1.7 µL of serum following immunodepletion. Therefore, the amount of intrinsic, aberrant TIMP1 isoform was estimated to be approximately 0.8 ng/mL serum. This demonstrates the sensitivity of the SISCAPA approach. The target TIMP1 peptide was also quantitated at a low (attomol) level from mass analyses of 2.5 µg of sample, although an increased relative error was observed (standard deviation, 14.6%). The quantitation of TIMP1 in this experiment was based on the assumption that aberrant glycoforms are quantitatively and selectively enriched from serum samples by L-PHA, and thus, it should be noted that the concentration of the aberrant TIMP1 in serum may actually be different from that estimated in this experiment. It is known that concentrations of intrinsic TIMP1 protein range in the hundreds of ng/mL and vary considerably in CRC serum.19 With the use of the coupling approach of L-PHAenrichment and the SISCAPA with MRM mass spectrometry, the aberrant TIMP1 present in a substoichiometric amount in colon cancer serum was analyzed quantitatively at a level of hundreds of pg/mL serum. This approach may be applicable as an analytical tool for understanding the biological function of a glycoprotein as it pertains to its glycan heterogeneity.

Conclusions Quantitative analysis of an aberrantly GlcNAcylated and substoichiometric isoform of the TIMP1 CRC serum glycoprotein at very low concentrations was accomplished by coupling L-PHA lectin enrichment with a SISCAPA protocol. L-PHA lectin selectively binds the β-1,6-GlcNAcylated glycoform, resulting in a much less complex solution of enriched glycoproteins. This enrichment step is crucial for the detection and quantitation of substoichiometric isoforms. The SISCAPA approach using tryptic digests of the resulting lectin-enriched glycoproteins provided for immunoenrichment of the analyte via anti-peptide antibody and allowed highly sensitive detection and quantitation via MRM mass analysis. With this combined technique, the aberrant TIMP1 was quantified at a level of 0.8 ng/mL serum. Thus, it was possible to perform highly sensitive and quantitative analysis of the aberrantly GlcNAcylated substoichiometric TIMP1 isoform present in sample prepared from human serum. This method, capable of selective quantitation of a specific glycoform from a variety of isoforms, is attractive for studies of glycan variability in proteins and may be further applicable to the discovery and validation of glycoprotein biomarkers. Journal of Proteome Research • Vol. 8, No. 9, 2009 4223

research articles Abbreviations: TIMP1, tissue inhibitor of metalloproteinase 1; β-1,6-GlcNAc, β-1,6-N-acetylglucosamine; GnT-V, N-acetylglucosaminyltransferase-V; CRC, colorectal cancer; L-PHA, phytohemagglutinin-L4; SISCAPA, stable isotope standards and capture by anti-peptide antibodies; MRM, multiple reaction monitoring; TLCK, N-R-Tosyl-Lys chloromethyl ketone.

Acknowledgment. This work was supported by the ‘Leading Foreign Research Institute Recruitment Program’ and ‘Convergence Research Center Program’ of Ministry of Education, Science and Technology. Supporting Information Available: Supplement 1, preparation of antibody. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Orntoft, T. F.; Vestergaard, E. M. Clinical aspects of altered glycosylation of glycoproteins in cancer. Electrophoresis 1999, 20, 362–371. (2) Hakomori, S. Inaugural Article: The glycosynapse. Proc. Natl. Acad. Sci. U.S.A 2002, 99, 225–232. (3) Choudhury, A.; Moniaux, N.; Ulrich, A. B.; Schmied, B. M. MUC4 mucin expression in human pancreatic tumours is affected by organ environment: the possible role of TGFbeta2. Br. J. Cancer 2004, 90, 657–664. (4) Dennis, J. W.; Laferte, S.; Waghorne, C.; Breitman, M. L.; Kerbel, R. S. Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 1987, 236, 582–585. (5) Barrabe´s, S.; Page`s-Pons, L.; Radcliffe, C. M.; Tabare´s, G.; Fort, E.; Royle, L.; Harvey, D. J.; Moenner, M.; Dwek, R. A.; Rudd, P. M.; De Llorens, R.; Peracaula, R. Glycosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer. Glycobiology 2007, 17, 388– 400. (6) Zhao, J.; Qiu, W.; Simeone, D. M.; Lubman, D. M. N-linked glycosylation profiling of pancreatic cancer serum using capillary liquid phase separation coupled with mass spectrometric analysis. J. Proteome Res. 2007, 6, 1126–1138. (7) Wulfkuhle, J. D.; Liotta, L. A.; Petricoin, E. F. Proteomic applications for the early detection of cancer. Nat. Rev. Cancer 2003, 3, 267– 275. (8) Ihara, S.; Miyoshi, E.; Ko, J. H.; Murata, K.; Nakahara, S.; Honke, K.; Dickson, R. B.; Lin, C. Y.; Taniguchi, N. Prometastatic effect of Nacetylglucosaminyltransferase V is due to modification and stabilization of active matriptase by adding beta 1-6 GlcNAc branching. J. Biol. Chem. 2002, 277, 16960–16967.

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