title: Identification of low-abundance cancer biomarker candidate TIMP1 from serum with lectin fractionation and peptide affinity enrichment by ultrahigh-resolution mass spectrometry.
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-L(4) (L-PHA) lectin, which shows binding affinity to the β-1,6-N-acetylglucosamine 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.
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, Ochang-Myun, Cheongwon-Gun, Republic of Korea.
authors.forename: Yeong Hee
authors.initials: YH
authors.lastname: Ahn
authors.forename: Kwang Hoe
authors.initials: KH
authors.lastname: Kim
authors.forename: Park Min
authors.initials: PM
authors.lastname: Shin
authors.forename: Eun Sun
authors.initials: ES
authors.lastname: Ji
authors.forename: Hoguen
authors.initials: H
authors.lastname: Kim
authors.forename: Jong Shin
authors.initials: JS
authors.lastname: Yoo
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D014408:Biomarkers, Tumor / Q000097:blood* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D015179:Colorectal Neoplasms / Q000175:diagnosis; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010835:Phytohemagglutinins / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000097:blood* / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D014408:Biomarkers, Tumor; D010455:Peptides; D010835:Phytohemagglutinins; D019715:Tissue Inhibitor of Metalloproteinase-1; C064103:phytohemagglutinin L4