The usefulness of multiple markers for diagnosis, prognosis and for predicting the risk of developing diseases or their complications is now widely recognized [13, 28–30]. Various proteomic approaches have been applied to biomarker discovery using biological fluids.
Regarding the search of biomarkers in CRC patients, several studies have been reported. The use of patient tissue samples allows comparison of the tumor sample with adjacent non-involved tissue. Alteration of 18 proteins was identified during tumor formation . Several other comparative proteomic studies of human CRC and adjacent normal tissue identified more proteins that appear to change consistently among patients with CRC [32–35]. Previously, our group identified gelsolin protein down-regulated in CRC . However, sample sizes are frequently limiting as priority has to be given to the use of surgically obtained tissue for conventional diagnosis .
Proteomic profiling is based upon the fact that proteins represent the dynamic state of the cells, reflecting earlier pathological and physiological changes in the disease more accurately than genomic sequencing . Proteomic patterns should assist in the detection of tumor biomarkers, as well as in evaluating the efficacy of anticancer drugs. The comparison of sera of cancer patients and healthy volunteers provided peaks from three differentially detected proteins, complement component 3a des-Arg, alpha-1-antitrypsin and transferrin, with 95% sensitivity and 91% specificity . Another SELDI study identified protein peaks that could distinguish CRC patients from normal subjects and classify patients with tumors at different stages, with 95% sensitivity and specificity . Our group identified protein peaks that could different lymph node positive CRC patients from negative ones . There has also been an effort to identify putative biomarkers in urine samples of CRC patients, with 19 protein peaks showing different intensities from those of healthy individuals (78% sensitivity, 87% specificity) . Further refinement and verification of these studies may eventually provide sets of clinically useful markers. Unfortunately, the proteome associated with CRC early diagnosis is currently poorly understood. Wang et al. identified two serum protein biomarkers (m/z 3961 and 5200 Da) for monitoring CRC . Previously, an extensive proteomic analysis in the serum of patients with CRC was performed. A standardized serum preparation method for MALDI-TOF-MS was utilized based on WCX MB and was able to identify many valuable, low-abundance protein masses of interest . However neither a characteristic protein cluster nor the structure identification of any of the proteins of the cluster was provided. Very recently it has been shown that clinical proteomic experiments can be still useful even when they deal with very small sample size .
In present study, a case control comparative analysis between CRC and health volunteers was performed by integrating the purification of proteins/peptides with WCX-MB, detection of peak intensity with MALDI-TOF MS, and profile analysis with ClinProt Tool software 2.2. Compared to controls, CRC shares 24 significantly differentiated proteins/peptides, including 9 up-regulated and 15 down-regulated peptides. By using the GA analysis, a cluster of 6 peptides at m/z 1208, 1467, 1505, 1618, 1656 and 4215 were developed as a classification mode, achieved a recognition capacity and a cross-validation of close to 100% to discriminate CRC from health volunteers. Also the classification mode could correctly classify CRC from health volunteers in blinded verification test, which surpass that of CEA.
The diagnostic capability of each peak at m/z 1208, 1467, 1505, 1618, 1656 and 4215 determined by ROC curve shows to be a highly accurate test (AUC> 0.88) . These protein/peptide fragments with high specificity and sensitivity may be good serum biomarkers for CRC. Later studies in a larger population group are necessary to confirm this finding. Further evaluation identified the 1505 and 1618 Da marker as alpha-2-HS-glycoprotein precursor and tubulin beta chain, respectively by LTQ Obitrap XL.
Alpha-2-HS-glycoprotein precursor, a single chain form of alpha-2-HS-glycoprotein, is converged into the two chain form after the completion of carbohydrate side-chain processing . Human serum protein alpha −2-HS glycoprotein is the human species homologue of bovine fetuin-A . Alpha-2-HS-glycoprotein is a circulating plasma glycoprotein, produced abundantly during fetal development by multiple tissues, whereas in the adult, it is produced predominantly by the liver . A number of studies suggest that alpha-2-HS-glycoprotein is a multifunctional protein . Difference expression of alpha-2-HS-glycoprotein exists in breast cancer, CRC, lung cancer, liver cancer, head and neck cancer [48–54]. As for CRC, serum alpha-2-HS-glycoprotein inhibits tumor progression by blocking transforming growth factor-β1 (TGF-β1) binding to cell surface receptors, suppressing TGF-βsignal transduction, and inhibiting TGF-β-induced epithelial-mesenchymal transition . Familial adenomatous polyposis (FAP) is one of the most important clinical hereditary forms of inherited susceptibility to CRC and is characterized by a high degree of phenotypic heterogeneity. Alpha −2-HS-glycoprotein was down-regulated in carpeting versus diffuse FAP patients and healthy donors, which was identified as serum molecule differently expressed in FAP patients . Increasing chronological age is a risk factor for many types of cancer including colorectal. An understanding of the biology of aging and factors which regulate it may provide insight into cancer pathogenesis. Maxwell F et al. identified that decreasing alpha-2-HS-glycoprotein concentration was associated with increasing chronological age, which indicates accelerated biological aging. Furthermore, alpha-2-HS-glycoprotein levels can be used to distinguish between colon and rectal cancers . However, serum alpha-2-HS-glycoprotein level was found not to be significantly changed by an ELISA test in CRC by another group . Presently, up-regulated alpha-2-HS-glycoprotein precursor was determined in CRC, which may perform as a diagnosis marker for CRC.
Tubulin, the subunit protein of microtubules, has generally been thought to be exclusively a cytoplasmic protein in higher eukaryotes. It’s an important target for anti-tumor drugs . Structurally, tubulin is an α/β heterodimer . Abnormal expression of the specific β-tubulin isotype is related to resistance to chemotherapy in solid cancers [59–61]. This marker may assume a greater role in therapeutic decision making. However, overexpression of subtypes of β-tubulin, class III β-tubulin and class VI β-tubulin, is a purely prognostic factor related to biologic aggressiveness of CRC, surprisingly this phenomenon is restricted to female patients . Early detection of malignancies of the gastrointestinal tract can lead to improved survival of patients worldwide. Beta-tubulin is a cancer-specific antigen in patients with CRC and other gastrointestinal malignancies, including gastric cancer, esophageal cancer and pancreatic cancer. Sensitivities ranged between 20% and 40% for various cancers with a specificity of 96% . We identified that β-tubulin up-regulated in CRC, which show great potential for diagnosis for CRC.
In conclusion, this study is one of the few study to screen CRC related proteins/peptides in sera by combining WCX-MB and MALDI-TOF-MS according to our knowledge. The classification model we have set up have application in providing alternatives for CRC diagnosis, and the up-regulated alpha-2-HS-glycoprotein precursor and β-tubulin provide a better understanding of the pathogenesis in CRC or help in tailoring the use of chemotherapy to each patient, finally resulting in an improvement in patient outcome. Continuing research into the underlying pathophysiology of CRC will ultimately lead to more effective and better-tolerated therapies. Additional analysis of a larger set of individual samples in combination with more traditional immunoassays such as ELISA are required to further confirm whether high level of serum alpha-2-HS-glycoprotein precursor and β-tubulin increased odds ratios (ORs) of CRC in a nested case–control sample of CRC individuals such as those observed in this study.