**3.1.4 Cervical carcinoma**

The serum peptidomics technology has been used in the research of endometrial carcinoma and is of guidance significance for the early diagnosis and clinical grading of diseases.

Our researches using the ClinTOF system have indicated that in the cervical carcinoma group and the control group, the protein peak intensify difference of only 21 proteins had statistical significance (P<0.05). M1450.35Da, M1778.7Da, M1896.65Da and M5520.42Da were taken as the classified variables to build a classification predictive model, to make classification diagnosis over the cervical carcinoma group and the control group, with the identification rate and predictive ability of 90.45% and 81.75% respectively. By contrasting the proteome of different pathological differentiation degrees, it is discovered that the protein peak intensity difference of 2 proteins had statistical significance (P<0.05). M5904.14Da and M5264.26Da were taken as the classified variables to build a classification model, to make classification diagnosis on different differentiation degrees, with the identification rate and predictive ability of 81.48% and 78.89% respectively.

Yoshizaki et al. adopted the SELDI system to analyze the protein profiling of 19 endometrial carcinoma cases and 20 normal tunica intima tissues, discovering two differential proteins, namely EC1 and EC2. The former had overexpression in the endometrial carcinoma tissues and the latter on the contrary. These differential proteins may be used for diagnosing endometrial carcinoma hopefully (Yoshizaki et al., 2005).

### **3.1.5 Leukemia**

The treatment of acute leukemia (AL) depends on minimal residual disease (MRD) diagnosis and detection. However there has been no serum biomarker that can be used by the clinicians for diagnosing AL and evaluating MRD. Yang et al. analyzed serum of AL patients, discovering two peptides (m/z of 1778 and 1865) reduced with the rise in the modification degree and that 1865 was related to AL types. After further FT-ICR-MS

Serum Peptidomics 385

with lymphocyte, neutrophilic granulocyte and monocyte and played a vital role in regulating host defense. Complement factor H is an important complement modulation substance and can also work as a significant effector molecule of congenital immunity. It is capable of distinguishing self and non-self and the activated and non-activated cell surfaces. In the research, the overexpression of two proteins indicates the inflammatory reaction, oxidation stress and immunologic mechanism are participating in the onset process of AD (Zhang et al., 2004). Hye et al. made case-control study to the plasma of 50 AD patients and the control group, discovering many disease pathology related proteins, including complement factor H (CFH) precursor and α22 macroglobulin and pointing out that the expression of CFH is related to the cognitive dysfunction of AD patients. α22 macroglobulin (A2M) is a pan-protease inhibitor. In case of brain damages, the immunological activity of A2M of the neuron and colloid cells will rise, especially at the levels of senile plaques, nerve

We utilized the SPE-C magnetic beads and ClinTOF system to compare 85 two-way patients having no medicine and 100 healthy people, discovering 5 significantly differential peaks in the mass range of 800Da-12,000Da among the mental disease patients and the healthy people. The serum polypeptide MS standard model profiling has a predictive sensitivity of

To study relevancy between some disease and polypeptide, we introduced serum polypeptide profiling into health screening. The ClinTOF system was used to study 1980 healthy people related polypeptide profiling researches, covering the age of 18-75, large sample statistics of both sexes, with the experimental design work shown in Fig.11. With

Fig. 11. Experimental Design of Healthy People Screening with the ClinTOF System

fiber matting and auantic neuron neurite (Hye et al., 2006).

**3.3 Healthy people screening** 

100% for the healthy people and for the mental disease patients, 87%.

detection, the two peptides are the C3F fragment. The linear regression analysis has indicated that the combined use of the peptides could distinguish PML / RARα positive molecules from M3. Findings show that the two C3F fragments are significantly correlated with the MRD level and can be used for evaluating clinical MRD (Liang et al., 2010).

#### **3.1.6 Brain glioma**

Our forward-looking researches on brain spongiocytoma diagnosis have indicated: using the SPE-C magnetic bead separation and liquid automatic processing robot operations of the ClinTOF system, after MS, 74 differential peaks occurred in the serum of 84 patients and 72 healthy people. 55 samples were taken randomly to build the MS model of patients and healthy people, with the diagnosis specificity for the healthy people reaching as high as 96.4% (Fig.10).

Fig. 10. Differential Polypeptide Peaks of Brain Glioma

#### **3.2 Nerve and mental diseases**

Alzheimer disease (AD) is a degenerative disease with unclear causes and mainly disturbing cerebral cortex neurons. Zhang et al. discovered that in the serum of AD patients, the haptoglobin and complement factor H were overexpressed. Haptoglobin is a kind of acute phase reactive protein and an antioxidant, featuring multiple biological activities and is considered to have participated in the inflammation process together

detection, the two peptides are the C3F fragment. The linear regression analysis has indicated that the combined use of the peptides could distinguish PML / RARα positive molecules from M3. Findings show that the two C3F fragments are significantly correlated

Our forward-looking researches on brain spongiocytoma diagnosis have indicated: using the SPE-C magnetic bead separation and liquid automatic processing robot operations of the ClinTOF system, after MS, 74 differential peaks occurred in the serum of 84 patients and 72 healthy people. 55 samples were taken randomly to build the MS model of patients and healthy people, with the diagnosis specificity for the healthy people reaching as high as

Alzheimer disease (AD) is a degenerative disease with unclear causes and mainly disturbing cerebral cortex neurons. Zhang et al. discovered that in the serum of AD patients, the haptoglobin and complement factor H were overexpressed. Haptoglobin is a kind of acute phase reactive protein and an antioxidant, featuring multiple biological activities and is considered to have participated in the inflammation process together

4940 4960 4980 5000 5020 5040 5060 5080 5100

m/z 5066.40

with the MRD level and can be used for evaluating clinical MRD (Liang et al., 2010).

Fig. 10. Differential Polypeptide Peaks of Brain Glioma

Disease Normal

**3.2 Nerve and mental diseases** 

 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 [arb. u.]

**3.1.6 Brain glioma** 

96.4% (Fig.10).

with lymphocyte, neutrophilic granulocyte and monocyte and played a vital role in regulating host defense. Complement factor H is an important complement modulation substance and can also work as a significant effector molecule of congenital immunity. It is capable of distinguishing self and non-self and the activated and non-activated cell surfaces. In the research, the overexpression of two proteins indicates the inflammatory reaction, oxidation stress and immunologic mechanism are participating in the onset process of AD (Zhang et al., 2004). Hye et al. made case-control study to the plasma of 50 AD patients and the control group, discovering many disease pathology related proteins, including complement factor H (CFH) precursor and α22 macroglobulin and pointing out that the expression of CFH is related to the cognitive dysfunction of AD patients. α22 macroglobulin (A2M) is a pan-protease inhibitor. In case of brain damages, the immunological activity of A2M of the neuron and colloid cells will rise, especially at the levels of senile plaques, nerve fiber matting and auantic neuron neurite (Hye et al., 2006).

We utilized the SPE-C magnetic beads and ClinTOF system to compare 85 two-way patients having no medicine and 100 healthy people, discovering 5 significantly differential peaks in the mass range of 800Da-12,000Da among the mental disease patients and the healthy people. The serum polypeptide MS standard model profiling has a predictive sensitivity of 100% for the healthy people and for the mental disease patients, 87%.

### **3.3 Healthy people screening**

To study relevancy between some disease and polypeptide, we introduced serum polypeptide profiling into health screening. The ClinTOF system was used to study 1980 healthy people related polypeptide profiling researches, covering the age of 18-75, large sample statistics of both sexes, with the experimental design work shown in Fig.11. With

Fig. 11. Experimental Design of Healthy People Screening with the ClinTOF System

Serum Peptidomics 387

We have used the ClinTOF system to study fetal congenital aplasia, with the research protocol shown in Fig.13. This thinking has generalized the sample collection strategies and the result expectations sufficiently indicating the diversification and universality of serum peptide method, and can be used for most research areas. Secretion leukoprotease inhibitory factor (SLPI) is an endogenic immunity related protein, capable of specifically inhibiting elastolytic enzyme, cathepsin G, trypsin and fibrinolysin and boasts of antiphlogistic response and antibiosis/antiviral activity. The detection of the content of SLPI protein in serum may have great clinical significance for diagnosis. Differential polypeptides were

obtained (Fig.14) and their functions were explained (Table 4).

Fig. 13. Research Protocol for **Fetal Congenital Aplasia** with ClinTOF System

**3.4 Fetal congenital aplasia** 

the SPE-C magnetic bead, MS profiling (Fig.12) for age 5 ranges was built and 10 peaks with the most significantly mass-to-charge ratio were identified. The online search of database with Mascot indicated that most polypeptide protein precursors were isogenous with the human prokinin precursors. After the analysis of serum peptidome profiling and bioinformatics data mining of 1890 healthy people, for the healthy people, the profiling has no obvious difference regardless of age and sex, but for people younger than 30, the sex difference factor shall be considered, that is, the elder the person is, the more different the serum peptidome profiling will be. Therefore, in researching the disease peptidome profiling, the age and sex matching factors shall be considered. It has also discovered that 50 polypeptides have a higher frequency of occurrence (more than 30%) in the serum of healthy people, indicating that these polypeptides can work as reference for health controls.

A: average profiling of 1890 healthy people; B and C: results from PCA analysis by age and sex; D: some peaks of highly-frequent occurrence detected by MS after the treatment by SPE-C magnetic beads

Fig. 12. Results of Healthy People Screening with the ClinTOF System

the SPE-C magnetic bead, MS profiling (Fig.12) for age 5 ranges was built and 10 peaks with the most significantly mass-to-charge ratio were identified. The online search of database with Mascot indicated that most polypeptide protein precursors were isogenous with the human prokinin precursors. After the analysis of serum peptidome profiling and bioinformatics data mining of 1890 healthy people, for the healthy people, the profiling has no obvious difference regardless of age and sex, but for people younger than 30, the sex difference factor shall be considered, that is, the elder the person is, the more different the serum peptidome profiling will be. Therefore, in researching the disease peptidome profiling, the age and sex matching factors shall be considered. It has also discovered that 50 polypeptides have a higher frequency of occurrence (more than 30%) in the serum of healthy people, indicating that these polypeptides can work as reference for health controls.

A: average profiling of 1890 healthy people; B and C: results from PCA analysis by age and sex; D: some peaks of highly-frequent occurrence detected by MS after the treatment by SPE-C magnetic beads

Fig. 12. Results of Healthy People Screening with the ClinTOF System

#### **3.4 Fetal congenital aplasia**

We have used the ClinTOF system to study fetal congenital aplasia, with the research protocol shown in Fig.13. This thinking has generalized the sample collection strategies and the result expectations sufficiently indicating the diversification and universality of serum peptide method, and can be used for most research areas. Secretion leukoprotease inhibitory factor (SLPI) is an endogenic immunity related protein, capable of specifically inhibiting elastolytic enzyme, cathepsin G, trypsin and fibrinolysin and boasts of antiphlogistic response and antibiosis/antiviral activity. The detection of the content of SLPI protein in serum may have great clinical significance for diagnosis. Differential polypeptides were obtained (Fig.14) and their functions were explained (Table 4).

#### Fig. 13. Research Protocol for **Fetal Congenital Aplasia** with ClinTOF System

Serum Peptidomics 389

Besides diseases studied by the ClinTOF system above, for the moment, we have finished the detection of near 10000 samples. Diseases are not only restricted to tumors, but covering

Stomach cancer 117 Fudan University, Shanghai Ruijin Hospital Esophagus cancer 48 West China University of Medical Science, CICAMS

Breast cancer 156 Beijing Cancer Hospital, Xinjiang Medical

Pancreatic cancer 120 Guangzhou People's Hospital

Leukemia 103 Shanghai Fifth Hospital

Endometriosis uterine 137 Beijing People's Hospital

Athletes 1995 PLA 301 Hospital

Diabetes 188 Shanghai Changzheng Hospital Test-tube baby tracking 102 Beijing University Third Hospital Mental disease 1220 Chongqing Medical University Healthy people 2473 Capital Medical University

Table 5. Diseases and Case Load Applied by the ClinTOF System (alterable)

Clinically, the high incidence and mortality rate of complex diseases like tumors cry for the new early-diagnosis patterns and effective early-diagnosis markers. In the mining of the mass spectrometric data, the cluster analysis and classification analysis have been used, but many problems still need further studies. In particular, the pretreatment of MS profiling, there is no standard method available. These issues are critical for putting serum peptidome into clinical detection. Secondly, along with the instant development and application of

University

Intestinal cancer 185 Shanghai Jiaotong University, Capital Medical University

Cognitive dysfunction 230 Zhongnan Hospital of Wuhan University,

75 Shenzhen People's Hospital

1678 PLA 301 Hospital, Guangzhou People's Hospital, Xian Jiaotong University, Hebei Medical

University Fourth Hospital

238 Sun Yat-Sen University Cancer Center

283 Beijing Obstetrics and Gynecology Hospital, Capital Medical University

303 PLA 301 Hospital, Tianjin Third Hospital

Shanghai Jiaotong University

almost all clinical aspects, as shown in Table 5.

Lung cancer, lung squamous carcinoma, adenocarcinoma of lung

Nasopharyngeal carcinoma

Rheumatosis, lupus erythematosus

**4. Prospect** 

Cirrhosis, hepatic carcinoma, hepatitis B

Endometrial carcinoma Endometrial carcinoma

**Disease Case Load Source** 

A and B: gel image of differential polypeptides



Table 4. Retrieval Results from Swiss-Prot Protein Database

chromosome group Normal

chromosome group

Normal chromosome group

Abnormal chromosome group

Abnormal chromosome group

Abnormal chromosome group

Normal

Abnormal chromosome group

Normal chromosome group

Fig. 14. Findings of Rsearching **Fetal Congenital Aplasia** with the ClinTOF System

A and B: gel image of differential polypeptides

Intensity

A

Intensity

B

Table 4. Retrieval Results from Swiss-Prot Protein Database

Besides diseases studied by the ClinTOF system above, for the moment, we have finished the detection of near 10000 samples. Diseases are not only restricted to tumors, but covering almost all clinical aspects, as shown in Table 5.


Table 5. Diseases and Case Load Applied by the ClinTOF System (alterable)
