**1.2.2 The 2nd generation of serum peptidomics research technology system: ClinProt**

To overcome the defects of SELDI, the ClinProt platform with functional magnetic beads, AnchorChip technology, MALDI-TOF-MS and ClinProTools has been developed. This platform has magnetic bead separating system, MS detecting system, analysis software and optional body fluid sample automatic processing system. The basic procedure is as follows: first of all, samples like serum, plasma, urine and cerebrospinal fluid, etc. from patients or the healthy controls have their high-abundance proteins and other impurities like salts to be eliminated and have the low-abundance proteins to be enriched with the magnetic bead. After separation and purification, samples are mixed with matrixes and directly drip on the AnchorChip target plate, and then protein mass spectrogram can be obtained through timeof-flight-MS (TOF-MS). The analysis software is employed to compare the differential expressed proteins of the patients or the healthy controls to get the specific mass spectra of the both for predicting the category of unknown samples (sick or healthy). Therefore, the core of this platform is the magnetic bead system and the software system.

Magnetic reagents include magnetic beads based Weak Cation Exchange (MB-WCX), Immobilized Metal Ion Affinity Chromatography Cu (MB-IMAC Cu) and Reverse Phase C18 (MB-RPC18), etc. The principle of magnetic beads is that the target proteins firstly combine with the magnetic microsphere surface ligand reversibility; secondly they will move in an outer magnetic field with a set direction by utilizing the magnetism of the magnetic beads to rapidly separate from surrounding medium. Compared with conventional technologies, the features of magnetic beads are: with large total surface area of magnetic beads, the specific basic groups on the surface can sufficiently combine with the low-abundance proteins in the serum to enhance the varieties of proteins, thereby to ensure the favorable specificity of the system; with simple and rapid operation, preliminary treatment can be finished through the simple blending, washing and elution process, which is suitable for clinical examination; the magnetic bead system has very high repetitiveness due to its large surface area; the liquid automatic processing system is a high throughput platform, it is capable to handle as many as 30,000 samples per day.

ClinProTools is a bioinformatics software boasting of all functions of biomarker detection and evaluation and capable of performing pre-processing of data, obtaining powerful, intuitive and visualized data from massive sample groups, carrying out peak statistics and pattern recognition, cluster analysis and independent test of data sensitivity, cross validation of specificity and meeting the classification demand of unknown samples.

The data processing of serum protein profiles commonly includes peak detection, data grouping, marker selection, marker evaluation and composite mode building. In information analysis and processing, two algorithms will be used: (1) non-monitoring algorithm, like self-organizing cluster analysis; (2) monitoring algorithm, like artificial neural network (ANN). These two, especially the latter one, are more and more found in

SELDI, however, has some disadvantages: it is incapable of identifying differential proteins screened online; in the profiling, the peak height and protein concentration not always have a linear relationship; in the detection process, many factors are involved but the control method hasn't been standardized yet, which leads to poor reproduction quality of

**1.2.2 The 2nd generation of serum peptidomics research technology system: ClinProt**  To overcome the defects of SELDI, the ClinProt platform with functional magnetic beads, AnchorChip technology, MALDI-TOF-MS and ClinProTools has been developed. This platform has magnetic bead separating system, MS detecting system, analysis software and optional body fluid sample automatic processing system. The basic procedure is as follows: first of all, samples like serum, plasma, urine and cerebrospinal fluid, etc. from patients or the healthy controls have their high-abundance proteins and other impurities like salts to be eliminated and have the low-abundance proteins to be enriched with the magnetic bead. After separation and purification, samples are mixed with matrixes and directly drip on the AnchorChip target plate, and then protein mass spectrogram can be obtained through timeof-flight-MS (TOF-MS). The analysis software is employed to compare the differential expressed proteins of the patients or the healthy controls to get the specific mass spectra of the both for predicting the category of unknown samples (sick or healthy). Therefore, the

Magnetic reagents include magnetic beads based Weak Cation Exchange (MB-WCX), Immobilized Metal Ion Affinity Chromatography Cu (MB-IMAC Cu) and Reverse Phase C18 (MB-RPC18), etc. The principle of magnetic beads is that the target proteins firstly combine with the magnetic microsphere surface ligand reversibility; secondly they will move in an outer magnetic field with a set direction by utilizing the magnetism of the magnetic beads to rapidly separate from surrounding medium. Compared with conventional technologies, the features of magnetic beads are: with large total surface area of magnetic beads, the specific basic groups on the surface can sufficiently combine with the low-abundance proteins in the serum to enhance the varieties of proteins, thereby to ensure the favorable specificity of the system; with simple and rapid operation, preliminary treatment can be finished through the simple blending, washing and elution process, which is suitable for clinical examination; the magnetic bead system has very high repetitiveness due to its large surface area; the liquid automatic processing system is a high throughput

ClinProTools is a bioinformatics software boasting of all functions of biomarker detection and evaluation and capable of performing pre-processing of data, obtaining powerful, intuitive and visualized data from massive sample groups, carrying out peak statistics and pattern recognition, cluster analysis and independent test of data sensitivity, cross

validation of specificity and meeting the classification demand of unknown samples.

studies regarding tumor diagnosis with satisfying results.

characteristic peaks of the same disease among different researchers.

core of this platform is the magnetic bead system and the software system.

platform, it is capable to handle as many as 30,000 samples per day.

Compared with SELDI, the magnetic bead particles have a larger total surface area due to their shapes and have stronger combining capacity, higher sensitivity and accuracy, so they perfectly cater for the research requirement of serum small-molecular proteins. Furthermore, biomarkers combined with the magnetic beads can be eluted, so this technology is suitable for MS and satisfy the requirement of sequential analysis in the further study. ClinProt system is able to perform the sequence identification task for the differentially expressed polypeptides/proteins to clearly distinct whether they are known or not.
