| Gastric carcinoma (GC) and colorectal carcinoma (CRC) are two of the most commonly diagnosed and the leading cause of cancer death the world, as well as in China. The high incidence rates and mortality of both two cancers in China reflect the great risk to human healthy. The great majority of these cancers and deaths could be prevented by increasing the use of established screening tests. However, even more progress is possible by increasing access to and utilization of GC and CRC screening tests; currently, only half of people aged 50 or older, for whom screening is recommended, have received the recommended tests. Early colorectal cancer often has no symptoms, which is why screening is so important and also is why patients refuse the invasive examination, such as endoscope. Reasons cited by survey participants for not participating in gastroenterological cancer screening include lack of time, inconvenience, lack of interest, cost, fear of being diagnosed with cancer, embarrassment, and unpleasantness of the test.However, studies show that this method is the most sensitive for the detection of colorectal cancer or adenomatous polyps. Noninvasive examination with high sensitivity and specificity is required for gastroenterological cancer screening. Blood is the most ideal sample source as it can be collected easily with less reluctance. Detection of blood tumor biomarkers has been highlight for years. Although researchers have bent to these work, only few of tumor biomarkers used in clinic are of convinced efficiency, such as alpha fetoprotein(AFP) and prostate-specific antigen(PSA).Some biomarkers offer suggestion for gastroenterological carcinoma diagnosis is not powerful enough by themselves, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). Based on the mass technology advances in serum peptidome may indicate new direction of cancer biomarker detection.1 Methodology optimization and bias investigation of serum peptidome researchSerum peptidome contains a large number of physiologic and disease-related information and attracts researchers'eyeballs in finding disease-related polypeptide markers. Stable serum peptidome profiles with high quality are the cornerstone of serum peptidome research. However, the serum composition is extremely complex that can adversely affect its mass spectrometric profiling. Moreover, it has been widely believed that the composition and abundance of serum polypeptides would change ex vivo, which is closely related with the outside environment. It is related to biases of clinical and analytical chemistries, mass spectrometry, sample handling, and spectral analysis. For instance, blood collection tubes, clotting times and temperature, and the number of freeze-thaw cycles are all critically important as well as surface chemistries and MALDI sample crystallization and laser irradiation, which were all major sources of variation. In addition, spectral alignment appeared to be the most challenging in terms of signal processing. When aligned properly, however, the resulting datasets are comparable to those obtained by common gene expression analysis, enabling the use of existing software packages. Besides, we wanted to address whether parameters such as gender and age influence peptidome profiles as obtained using our mass spectrometry-based serum peptide profiling platform. In this paper, we studied comparatively in the numerous details of the process of the sampling and treatment of blood, the pretreatment of serum (magnetic beads and ultra filtrate centrifugal Filters), the mass spectrometry and the data handling, and analyzed the effectiveness of gender and age differences on the serum peptidome. Effectiveness of variables on serum peptidome research based on MB-WCX and MALDI-TOF mass spectrometry was unfolded. The optimal variables setting was obtained, which provide evidence for serum peptidome research. The results demonstrated that each step of the serum peptidome experiment should be standardized.2 Specific expression of serum peptidome and foundation of diagno -stic model on gastric cancer and colorectal cancerRecent years studies of serum peptidome show that the state of exception tumor cell growth, invasion, and the change of immune system along with the change of enzyme and protein hydrolysis process, can cause the body's serum peptidome specific changes. The early studies of breast cancer, prostate cancer, thyroid cancer and oral cancer show that one can get good diagnosis efficiency by building model of combined specific serum peptides. In this study, we employed weak cation exchange magnetic beads (MB-WCX) to desalt samples and remove abundant proteins. Serum peptidomic profiles from 40 GC patients and 40 CRC patients without treatment and 40 healthy controls were obtained by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The serum peptidome research platform has been standardized and optimized in previous research. Comparing the CRC group and control group with Wilcoxon test, we discovered that 81 peaks were of significant differences (p<0.00001) between the two, about 46.82% of the total serum peptides; 79 peaks of marked difference were picked out by comparing GC group with control group (p＜0.00001)。A novel strategy for selecting an optimal combination of key elements of multi-component spectral data, named competitive adaptive reweighted sampling (CARS), is adopted. Two optimal subsets of peaks were selects by using CARS method and pattern recognition equations were established through partial least squares-discriminant analysis (PLS-DA).One optimal subset of 5 peptide peaks (X1 3217.07m/z, X2 3890.71 m/z, X3 3972.74 m/z, X44963.99m/z和X5 5864.13m/z)selected by comparing CRC group and control group was used to set up a discriminant equation (Z=0.5733X1+0.4129X2+ 0.5388X3-0.4420X4-0.4899X5) to distinguish CRC from normal with a 10-fold cross validation accuracy of 95.1%,a sensitivity of 89.9% and specificity of 98% in double blind test on test group. Similarly, a GC discriminant equation was established to distinguish GC from normal: Z=0.4835Y1+0.5390Y2+0.5189Y3-0.4614Y4-0.4317Y5 (Y,2311.37m/z, Y24210.40 m/z, Y35539.74 m/z, Y46005.97 m/z, Y56666.69m/z) 10-fold cross validation shows the accuracy of this equation in GC diagnosis is 95.1%.Meanwhile, the equation was evaluated by double blind test on test group, a sensitivity of 89.9%and a specificity of 98% was acquired. The findings show that significant differences exist among serum peptide expression profiles of GC patients, CRC patients and normal controls.The tumor-specific expression patterns of serum peptide has the potential of gastrointestinal tumor markers. This study is based on strict standardized and optimized experimental platform, to take full advantage of new statistical means to filter out a subset variable. The discriminant equations can more accurately distinguish cancer patients with normal human; significantly improve the diagnostic efficiency of GC and CRC. This work provides researchers a new way to find novel tumor specific serum markers and to develop a simple but non-invasive cancers screening method. |
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