Identification of Low-Abundance Cancer Biomarker Candidate TIMP1

Article pubs.acs.org/ac

Identification of Low-Abundance Cancer Biomarker Candidate TIMP1 from Serum with Lectin Fractionation and Peptide Affinity Enrichment by Ultrahigh-Resolution Mass Spectrometry Yeong Hee Ahn,† Kwang Hoe Kim,†,‡ Park Min Shin,† Eun Sun Ji,† Hoguen Kim,§ and Jong Shin Yoo*,†,‡ †

Division of Mass Spectrometry, Korea Basic Science Institute, Ochang-Myun, Cheongwon-Gun, Republic of Korea GRAST, Chungnam National University, Daejeon, Republic of Korea § Department of Pathology, Yonsei University College of Medicine, Seoul, Republic of Korea ‡

ABSTRACT: As investigating a proteolytic target peptide originating from the tissue inhibitor of metalloproteinase 1 (TIMP1) known to be aberrantly glycosylated in patients with colorectal cancer (CRC), we first confirmed that TIMP1 is to be a CRC biomarker candidate in human serum. For this, we utilized matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) showing ultrahigh-resolution and high mass accuracy. This investigation used phytohemagglutinin-L4 (L-PHA) lectin, which shows binding affinity to the β-1,6-Nacetylglucosamine moiety of N-linked glycan on a protein, to compare fractionated aberrant protein glycoforms from both noncancerous control and CRC serum. Each lectin-captured fraction containing aberrant glycoforms of TIMP1 was digested by trypsin, resulting in the tryptic target peptide, representative of the serum glycoprotein TIMP1. The resulting target peptide was enriched using a stable isotope standard and capture by the antipeptide antibody (SISCAPA) technique and analyzed by a 15 T MALDI FTICR mass spectrometer with high mass accuracy (Δ < 0.5 ppm to the theoretical mass value of the target peptide). Since exact measurement of multiplex isotopic peaks of the target peptide could be accomplished by virtue of high mass resolution (Rs > 400 000), robust identification of the target peptide is only achievable with 15 T FTICR MS. Also, MALDI data obtained in this study showed that the L-PHA-captured glycoforms of TIMP1 were measured in the pooled CRC serum with about 5 times higher abundance than that in the noncancerous serum, and were further proved by MRM mass analysis. These results confirm that TIMP1 in human serum is a potent CRC biomarker candidate, demonstrating that ultrahigh-resolution MS can be a powerful tool toward identifying and verifying potential protein biomarker candidates.

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to fractionate protein glycoforms that have a specific glycan structure with binding affinity to lectin. Therefore, this approach is very attractive as a mode of sample preparation to examine the difference between protein glycoforms. A variety of lectins has been employed to fractionate specific protein glycoforms from complex glycoproteome. Mass spectrometry (MS) has emerged as a compelling means of gathering both qualitative and quantitative information regarding lectin-captured target proteins. MS is a powerful tool that has been extensively applied in the field of proteomics. Among the many types of MS, Fourier transform ion cyclotron resonance (FTICR) MS demonstrates high resolution (Rs > 400 000) as well as high mass accuracy (Δ < 0.5 ppm mass error).26−28 FTICR achieves its highest analytical performance when equipped with a electrospray ionization (ESI) interface for sample introduction. This setup

berrant carbohydrate modifications of the glycan moiety on glycoproteins may be closely associated with pathogenic processes occurring in cancer cells.1−5 Various aberrant glycosylation patterns have been connected to cancer. Many clinical cancer diagnostic tests employ glycoproteins, including prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), and CA-125.6−9 Thus, an aberrant change in protein glycosylation state is important in studying pathological mechanisms implicated in cancer or for developing cancer biomarkers. A common example of this is α-fetoprotein (AFP), known to be a potent biomarker for hepatocellular carcinoma (HCC). AFP-L3, an aberrantly fucosylated glycoform showing increased affinity to fucose-specific lectins like Lens culinaris agglutinin (LCA), has been reported to show improved specificity as a biomarker for HCC.10−12 Because of increasing interest in the biological relationship between cancer and specific protein glycosylation, a variety of enrichment methods for glycoproteins and glycopeptides has been investigated including lectin-fractionation,13−18 chemicalbased capturing,19,20 and chromatographic separation.21−25 Among them, the lectin-based approach has been widely used © 2011 American Chemical Society

Received: September 21, 2011 Accepted: December 23, 2011 Published: December 23, 2011 1425

dx.doi.org/10.1021/ac2024987 | Anal. Chem. 2012, 84, 1425−1431

Analytical Chemistry

Article

(bovine), β-casein (bovine), serum albumin (bovine), ovalbumin (chick), and myoglobin (horse) were obtained from Sigma-Aldrich (St. Louis, MO). Streptavidin magnetic bead (MB) conjugate and Dynabead MyOne tosylactivated MBs were obtained from Invitrogen (Carlsbad, CA). Biotinylated phytohemagglutinin-L4 (L-PHA) lectin was obtained from Vector Laboratories (Burlingame, CA). All other reagents were obtained from Sigma-Aldrich, unless noted otherwise. Serum Sample Preparation and Lectin Fractionation. CRC sera were obtained from patients with CRC at the Severance Medical Center at Yonsei University (Seoul, Korea); samples included in the study were used by permission of the patients within the study. A pooled CRC serum was prepared by mixing an equal amount of serum from five of the patients with CRC; as a noncancerous control serum sample, a pooled control serum was obtained from Sigma-Aldrich (product number H6914) prepared from male donors. Protein concentration of each pooled serum was quantified using Quant-iT protein assay kits (Invitrogen). Each pooled serum sample (320 μL) was diluted with 50 mM ammonium bicarbonate until the concentration of spiked CaCl2 and MgCl2 in the sample reached 5 mM. The serum solution then was mixed with L-PHA-immobilized MB (16 mg) and gently shook overnight at 10 °C. The immobilized lectin was prepared in advance by mixing biotinylated L-PHA with streptavidin−MB for 1 h at room temperature as per the manufacturer’s instructions. Prior to protein binding, the mixture was washed extensively with phosphate-buffered saline (PBS). Lectin-bound protein was washed three times with PBS and eluted using an elution buffer (2 M urea, 0.5 mM DTT, 50 mM ammonium biocarbonate). After cysteine blocking with iodoacetamide for 30 min in the dark, the eluted proteins were digested with trypsin (20 μg) overnight at 37 °C. Trypsin digestion was stopped by adding 1% formic acid. Samples were spiked with stable isotope-coded internal standard of the target peptide GFQALGDAADIR (heavy, 500 fmol), and tryptic digests were desalted using an Oasis HLB column (Waters, Maidstone, Kent, U.K.) and lyophilized in a SpeedVac system (Thermo Scientific, Wiesbaden, Germany). Immobilization of an Antipeptide Antibody on Magnetic Beads. Manufactured antipeptide antibodies were immobilized on tosylactivated MBs according to the manufacturer’s instructions. Briefly, the antibody was added to a suspension of MBs (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 10 min. After the addition of 3 M ammonium sulfate buffer, the reaction tube was slowly agitated at 37 °C for 16 h 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) bovine serum albumin (BSA) and 0.05% Tween 20], followed by slow agitation at 37 °C for 16 h. 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]. Antibody Enrichment of CRC Serum. Tryptic digests of the lectin-captured CRC serum were reconstituted in PBS buffer and mixed with the bead-immobilized antibody (500 μg beads) that had been prewashed with PBS (3 × 200 μL). The supernatant was removed after slow agitation at room temperature for 1 h. The beads were washed thoroughly with PBS (3 × 200 μL), and the enriched target peptides were

has been applied to mass profiling experiments for various proteome samples originating in biological media, although mass profiling data can only provide qualitative or semiquantitative information about the identified proteins. For quantitative analysis of target proteins identified by mass profiling experiments, a multiple reaction monitoring (MRM) method, usually operating on a triple quadrupole mass spectrometer, has been widely used.17−19,29 The MRM method can provide specificity and sensitivity sufficient to quantify an abundance of target peptides by virtue of two-staged mass selection as well as concurrent chromatographic elution of the target peptides and their stable isotope-coded internal standards. Recently, we reported a coupling approach, combining the comparative lectin-fractionation process and MRM-based analysis to quantify an abundance of aberrant glycoforms of target proteins, including tissue inhibitor of metalloproteinase 1 (TIMP1), in the samples prepared by the comparative lectinfractionation.18 The study differed in that the MRM-based assay was conducted with lectin-captured secretomes obtained from culture media of colorectal cancer (CRC) cell lines rather than with serum samples; despite this difference, this demonstrated that the lectin-coupled MRM-based approach could be an attractive tool to verify biomarker candidates involved in aberrant protein glycosylation. As compared to proteomes secreted from cell cultures under well restricted conditions, serum proteomes show high sample complexity, a wide dynamic range in protein abundance, and dramatic variation between individuals. Thus, if a target protein is present at low concentrations in the serum sample, affinity enrichment using an antibody can effectively concentrate the target protein or the target peptide as a surrogate of the target protein, reducing the sample complexity and improving the analytical performance of the mass analysis performed. This target peptide enrichment protocol, using a stable isotope standard and capture by an antipeptide antibody (SISCAPA) technique, is efficient as the enriched antigen (i.e., the target peptide) is directly subjected to mass analysis without further sample treatment such as proteolysis.17,30−33 In this study, an ultrahigh-resolution MS-based method for identifying and verifying an aberrantly glycosylated protein cancer biomarker candidate in human serum was developed. First, samples prepared from a pooled control and a pooled CRC serum by using a lectin-capturing and a peptide affinity enrichment (SISCAPA) technique were analyzed using a 15 T (15T) MALDI FTICR mass spectrometer. As the 15 T FTICR mass spectrometer has a sufficient baseline mass resolution such that each isotope peaks of the target peptide may be exactly identified, we expect that the robust identification for the target peptide in a complex proteome sample of human serum can be accomplished simply using MALDI MS.

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EXPERIMENTAL SECTION Materials. The synthetic peptide GFQALGDAADIR and its stable isotope-coded counterpart were obtained from Anygen Corporation (Kwangju, Korea). Mass analysis of the stable isotope-coded peptide GFQALGDAADIR (isotopically labeled site in italics; 13C and 15N incorporated) was identified by mass shifts of +4 Da. Monoclonal antibodie against the tryptic peptide GFQALGDAADIR was manufactured by ABFrontier (Seoul, Korea). Trypsin (sequencing grade) was purchased from Promega (Madison, WI). Standard proteins for preparation of a matrix peptide solution, including α-casein 1426



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eluted with an elution buffer (0.1 M glycine-HCl, pH 1.5) and dried using a SpeedVac system. The target peptide sample was reconstituted with 0.5% trifluoroacetic acid and desalted by Zip-TipC18 for MALDI mass analysis. MALDI Mass Analysis. Ultrahigh-resolution mass analysis of the target peptide enriched by SISCAPA was performed in broadband mode using a 15 T MALDI FTICR mass spectrometer (15 T Bruker Apex-Qe FTICR MS, 355 nm Nd:YAG laser; Bruker Daltonics, Bremen, Germany). The system interface utilized a Bruker ApexControl 2.0 system (Bruker Daltonics, Bremen, Germany). The peptide sample was loaded and crystallized onto stainless steel MALDI plates with α-cyano-4-hydroxycinnamic acid as the MALDI matrix. All shots were accumulated in broadband mode with a 1 megabyte time-domain signal. External calibration of the MALDI mass spectra using six standard peptide solutions demonstrated a mass accuracy of 0.3 ppm in the range of m/z 800−4000 on positive ion mode. The MS spectrum of each sample was reported by averaging 30 signals accumulated by 100 laser shots.

Figure 1. Workflow for mass analysis for aberrant glycoforms of the target protein in human serum by a lectin-coupled/SISCAPA approach.

of TIMP1 fractionated by L-PHA lectin from the healthy serum and the CRC serum was examined. Target Peptide Calibration and Immobilized Antibody Efficiency. Since the lectin-captured target protein TIMP1 may show deep dynamic concentration levels, linear concentration ranges and precision of the target peptide of TIMP1 in MALDI MS analysis were investigated using the synthetic target peptide (light, L) and its stable isotope-coded counterpart (heavy, H). As shown in Figure 2, good linearity

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RESULTS AND DISCUSSION Serum Sample Preparation by Lectin Fractionation/ SISCAPA Approach. Many serum proteins, including TIMP1, have been shown to experience aberrant glycosylidation linked with the progression of certain cancers. TIMP1, a target protein for GnT-V in colon cancer cells, is a glycoprotein with two Nglycan moieties at residues Asn53 and Asn101. It has been reported to be involved in the progression of certain types of cancer through abnormal glycosylation via the terminal addition of β-1,6-N-acetylglucosamine to the N-linked glycan of the protein in invasive/metastatic cancer cells.34 In our previous study, we showed that aberrant glycoforms of TIMP1 could be captured efficiently by L-PHA lectin, a lectin that specifically recognizes the β-1,6-N-acetylglucosamine moiety of the Nlinked glycan.17,18 To compare difference in the L-PHA-specific glycoforms of glycoproteins between control and CRC serum, we prepared a pair of samples, a pooled control serum and a pooled CRC serum. Because of difficulty in collecting serum sample of healthy individuals from medical center, we used a commercially available pooled serum as a control sample. Since the control sample to be compared with cancer sample was secured in the state of the pooled serum, the pooled cancer sample was also prepared by mixing cancer sera (each 100 μL) of five patients with CRC. Sample pooling may provide an additional advantage in biomarker identification stage by virtue of the averaging effect for unwanted biological variation between cancer patients, under the situation available only limited sample number. A pair of pooled serum samples prepared was fractionated in parallel using L-PHA lectin (Figure 1). L-PHA lectin immobilized via biotin−streptavidin conjugation to magnetic beads, rather than agarose beads with a polyhydroxy group on the agarose backbone, was employed to capture L-PHA-specific aberrant protein glycoforms. Each LPHA-captured fraction of the pooled control serum and the pooled CRC serum was digested by trypsin. As a representative of the target glycoprotein TIMP1, tryptic peptide GFQALGDAADIR (m/z 1233.6) was selected as the antigens for SISCAPA enrichment. The tryptic target peptide has been identified with relatively strong signal intensity and considered not to contain any residue of post-translational modification.17 By comparative MALDI mass analysis of the samples enriched by the SISCAPA technique, abundance of aberrant glycoforms

Figure 2. Linear concentration range for MALDI FTICR mass analysis for the target peptide (GFQALGDAADIR). Each MS measurement was conducted with an equal amount of stable isotope-coded internal standard (heavy, 100 fmol) and a different amount of target peptide (light, from 0.5 fmol to 1 pmol). Satisfactory concentration linearity and precision was observed over a concentration range spanning 3 orders of magnitude.

with respect to concentration (R2 = 0.9934) was observed spanning 3 orders of magnitude of concentration, while also demonstrating acceptable precision (CV < 9.6% for triplicate measurements of L/H). On the basis of the dynamic range in concentration, we evaluated the binding efficiency of the MB-immobilized antipeptide TIMP1 antibody. The antigen peptide (L) was spiked into a matrix peptide solution (prepared by trypsin digestion of standard proteins) and enriched using beadimmobilized antibodies (500 μg beads). After spiking the stable isotope-coded internal standard (H) with the enriched sample, the sample solution was analyzed by MALDI MS analysis. 1427



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by lectin and enriched by peptide antibodies, is assumed to be far lower than that of total glycoforms of TIMP1. MALDI MS Analysis of Aberrantly Glycosylated TIMP1 by Lectin-Capturing/SISCAPA. The purpose of this study was to establish an ultrahigh-resolution MS-based protocol using lectin specific fractionation and peptide affinity-based enrichment techniques for verifying a low-abundance cancer biomarker candidate. Additionally, this study aimed to confirm whether the target protein TIMP1 could be developed as a potent CRC serum biomarker. Thus, each pooled sample of CRC and control sera was fractionated using L-PHA lectin and subsequently trypsin-digested to give the tryptic target peptide as shown in Figure 1. Preliminary experimentation demonstrated that direct measurement of different TIMP1 glycoforms L-PHA-captured from a serum sample is difficult; thus, further enrichment of the target peptide surrogating the lectin-captured TIMP1 glycoforms was required.17 This preliminary experiment showed that enrichment of the target peptide from the very complex tryptic digest solution that results from the lectincaptured serum fraction could be accomplished efficiently using the SISCAPA technique, with high sensitivity detection of the enriched target peptide possible using LC−MRM mass analysis. In this study, the target TIMP1 lectin-captured peptide was further concentrated by applying the SISCAPA method to each of the tryptic digests of L-PHA-captured fractions from both control and CRC sera. Each SISCAPA-enriched sample was analyzed using the 15 T MALDI FTICR mass spectrometer

Table 1 shows binding efficiency of the bead-immobilized TIMP1 antibody (CV, 13.7% for triplicate experiments). The Table 1. Efficiency Tests of Magnetic Bead-Immobilized TIMP1 Anti-Peptide Antibodiesa

test 1 test 2 test 3 average CV (%)

spiked antigen (L, pmol)

spiked standard (H, pmol)

captured avg ratio (L/H)

CV (%)b

4 4 4

4 4 4

0.704 0.547 0.699

2.7 2.2 2.5

captured antigen (L, pmol)

antigen recovery (%)

2.815 2.189 2.797 2.600 13.7

70.4 54.7 69.9 65.0 13.7

a

Each mass measurement was conducted using a sample concentration corresponding to the internal standard (H), 100 fmol. bCV value for triplicate mass analyses in each test.

bead-immobilized antibody was capable of capturing up to a few pmol of the target peptide under the used enrichment condition. Previous investigations estimated the abundance of total TIMP1 in human blood samples to be about a few pmol per milliter.35 Aberrant glycoforms of TIMP1 are present in substoichiometric amounts to that of total TIMP1 because of microheterogeneity in protein glycosylation; because of this, the abundance of aberrant glycoforms of TIMP1, fractionated

Figure 3. MALDI FTICR MS spectra obtained from pooled control and CRC serum samples prepared by the L-PHA-capturing/SISCAPA method. Δ represents the ratio of mass error to theoretical mass value for each isotopic peak of the target peptide and its internal standard. Only the mass values corresponding to first and second monoisotopic mass peaks of each peptide detected were labeled on spectra for simplicity. 1428



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Table 2. Mass Values of the Target Peptide and Its Internal Standard Counterpart Measured for the Pooled Control and CRC Sera by 15 T MALDI FTICR MS target peptide (L)

internal standard (H)

sample

isotopic peaks

m/z (obsd)

m/z (theor)

Δ (ppm)

m/z (obsd)

m/z (theor)

Δ (ppm)

synthetic standard

first second first second first second

1233.6226 1234.6259 1233.6231 1234.6263 1233.6225 1234.6260

1233.6222 1234.6256 1233.6222 1234.6256 1233.6222 1234.6256

0.324 0.243 0.730 0.567 0.243 0.324

1237.6293 1238.6327 1237.6294 1238.6327 1237.6298 1238.6335

1237.6293 1238.6329 1237.6293 1238.6329 1237.6293 1238.6329

0.000 0.161 0.081 0.161 0.404 0.484

control serum CRC serum

using the isotopic peaks of a target peptide are another feature obtainable using ultrahigh-resolution MS. Thus, robust identification of the endogenous target peptide in complex tryptic digest solutions prepared from human sera could be accomplished by multiplex peptide mass confirmation using isotopic peaks of a target peptide. Many studies using high mass resolution of FTICR MS for peptide identification and database searching were reported.27,36−38 In peptide identification with a measured mass value, the number of candidate peptide enable to be matched to a database decreases with increasing of mass accuracy.39,40 However, it is still assumed that single matching to the measured mass value is impossible to accomplish with the mass accuracy (0.5 ppm) obtained by the broad band FTICR MS used in the study. Nonetheless, since the target peptide to be identified in this study was already enriched specifically by monoclonal antipeptide antibody, which can reduce dramatically the complexity of a sample mixture, more robust identification of the target peptide could be accomplished through using the ultrahigh resolution MALDI MS, even without using LC separation which is an inevitable process in MRM-based analysis. So we think the integrative using of the target peptide-specific antibody enrichment and ultrahighresolution broad band MALDI MS can be an attractive strategy for peptide biomarker identification and verification as shown for the serum cancer biomarker candidate TIMP1, without requiring a LC separation and MS/MS data. Confirmation by LC−MRM MS. The identification of glycoprotein TIMP1 as a serum cancer biomarker candidate by using ultrahigh-resolution MALDI FTICR MS was further confirmed by another analytical method using LC−MRM MS. The LC-coupled MRM method has been widely used for the quantitative analysis of target peptides originating from a complex proteome sample. In a previous study, we reported that the aberrant glycoforms of TIMP1 could be detected using LC−MRM MS from a CRC serum sample prepared by lectin fractionation/SISCAPA approach.17 Using samples prepared separately from both a control and CRC sera by L-PHA lectin fractionation/SISCAPA technique with a reduced preparation scale, LC−MRM mass analyses were conducted following the analytical conditions described in the previous paper.17 Figure 4 shows the extracted chromatograms obtained from MRM mass analyses of tryptic digests of the samples prepared from both control and CRC sera. By examining the chromatographic peak area ratios of the transition pairs selected for the target peptide and its internal standard counterpart, the difference in abundance of the target peptide in both the control and CRC serum samples was calculated (cancer/control, 0.066/ 0.016 = 4.1). The abundance of the target peptide measured with the CRC sample is higher 4.1 times than that with the control sample, which resembles the result (4.6 times) obtained

calibrated externally using a calibration mixture containing six peptides. Figure 3 shows the MALDI MS spectra obtained from each sample prepared using the lectin-capturing/SISCAPA method. In spectra from both control and CRC samples, the peaks corresponding to those spiked with internal standard (H) were detected at m/z 1237.6294 and 1237.6298, respectively, as the first monoisotopic mass peak. Additionally, the second monoisotopic peak appeared at m/z 1238.6327 and 1238.6335, respectively, within both spectra. Resolution of each isotopic peak corresponding to the internal standard was observed with complete baseline resolution utilizing the FTICR mass spectrometer. The difference between the two spectra, obtained, respectively, for the pooled control and the pooled CRC serum, was confirmed by observing the target endogenous peptide (L) within the serum samples. The target peptide TIMP1 was detected only with a trace signal at m/z 1233.6231 as the first monoisotopic peak of the target peptide in the spectrum obtained from the control serum sample; however, this target peptide was detected within the CRC serum sample at m/z 1233.6225 with a higher peak intensity, corresponding to 4.6 times of the peak intensity in the spectrum obtained from the control serum (avg cancer (L/H)/avg control (L/H), 0.401/0.087 = 4.6). Some ghost peaks were detected in the mass spectra, which may have been due to nonspecific binding occurring in the SISCAPA process. Note that ghost peaks at m/ z 1234.7061 and 1234.7064 were fully differentiated from second monoisotopic peaks of the target peptide at m/z 1234.6263 and 1234.6260 due to the ultrahigh-resolution capabilities of the FTICR mass spectrometer. These results show that despite the presence of some undesirable and unidentifiable ghost peaks from sample matrix, the robust identification of the target peptide originating from human serum as well as the discrimination target protein TIMP1 between cancerous and noncancerous serum could be easily accomplished using MALDI MS coupled with the lectin capture and SISCAPA enrichment. Multiplex Identification Using Isotopic Peaks of the Target Peptide. The observed mass values of the target peptide, as measured by 15 T MALDI FTICR mass analysis, is summarized in Table 2. Among the isotopic peaks of the target peptide (L) and its internal standard counterpart (H), only the first and second fine isotopic masses of each of the peaks normally detected are shown in Table 2. Errors associated with mass values of the target peptide and internal standard, as compared to the theoretical mass values calculated for these peptides, were within 0.5 ppm over all measured values in the study, except the mass values of the target peptide (L) measured at trace intensities with mass errors outside 0.5 ppm from the control serum sample. In addition to highly accurate peptide identification, multiplex peptide mass confirmation 1429



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solution via peptide affinity-based enrichment (SISCAPA) and analyzed by ultrahigh-resolution MALDI FTICR MS. Finally, the aberrant TIMP1 glycoform was confirmed to be a potent CRC biomarker candidate, opening the door to future biomarker validation for clinical study. Note that as the 15T MALDI FTICR mass spectrometer utilized shows ultrahigh mass resolution and mass accuracy such that isotope peaks of a target peptide may be exactly identified, and multiplex peptide isotopic mass confirmation could be accomplished normally with mass errors within 0.5 ppm. Thus, exact identification and quantitative analysis of a target peptide from complex serum media could be accomplished simply with its MALDI MS spectrum. The study presented above thus demonstrates that ultrahighresolution FTICR MS can be used as a practical tool for rapidly verifying biomarker candidates using a large number of samples.

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AUTHOR INFORMATION

Corresponding Author

*Address: Jong Shin Yoo, Division of Mass Spectrometry, Korea Basic Science Institute, 804-1 Yangcheong-Ri, OchangMyun, Cheongwon-Gun 363-883, Republic of Korea. Phone: +82-43-240-5150. Fax:+82-43-240-5159. E-mail: jongshin@ kbsi.re.kr. Figure 4. The extracted chromatograms obtained from the MRM mass analyses of samples prepared by the L-PHA-capturing/SISCAPA method from the control serum (a) and pooled CRC serum (b). The samples were prepared by using 100 μL of each serum and spiking the internal standard (1 pmol). The chromatograms were displayed for the transition pairs, the m/z 617.31 → 717.35 transition of the endogenous target peptide (light, GFQALGDAADIR) and the m/z 619.31 → 721.38 transition of the stable isotope-coded internal standard (heavy). The peaks gray-stained correspond to the target peptide and its internal standard. The abundance of the target peptide in both the control and CRC serum samples was calculated based on the chromatographic peak area ratios of the transition pairs selected for the target peptide and its internal standard counterpart.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by the Convergence Research Center Program (Grant Number 2011K000884) from the Korean Ministry of Education, Science, and Technology and by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (2009, University-Institute Cooperation Program).

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by using ultrahigh-resolution MALDI FTICR MS in this study. Thus the result obtained by LC−MRM MS confirmed that the ultrahigh-resolution MALDI MS could provide the reliable result in identifying the aberrant glycoprotein TIMP1 as a CRC biomarker candidate. This also means that the target glycoprotein TIMP1 is a potent CRC biomarker candidate; a further verification study using massive blood samples obtained from large cohorts is necessary, which will be the subject of a continued study.

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CONCLUSIONS Using a 15 T MALDI FTICR mass spectrometer, featuring high resolution and mass accuracy, ultrahigh-resolution MS was confirmed to be a powerful tool toward rapidly identifying peptide biomarkers in human serum, such as the CRC serum biomarker candidate TIMP1. To perform mass analysis of the protein cancer biomarker aberrantly glycosylated in patients with CRC, sample preparation was conducted using a lectinfractionation method to collect aberrant protein glycoforms having a specific glycan structure. Aberrant protein glycoforms fractionated by L-PHA lectin from samples originating either from pooled CRC or healthy serum were digested with trypsin, resulting in solutions containing the TIMP1 target peptide. The TIMP1 target peptide was further enriched from tryptic digest 1430



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Identification of Low-Abundance Cancer Biomarker Candidate TIMP1

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