**1.2.1 The 1st generation of serum peptidomics platform: SELDI**

Different from MALDI-TOF-MS, the surface enhanced laser desorption/ionization(SELDI) time of flight mass spectrometry platform combines the protein chip with multiple high technologies (highly integrated, ultra-micro, computerized, automat-zed) based on the chromatogram principle, thereby to strengthen the affinity and capture ability of the chip so that proteins are selectively absorbed by the chemically-modified solid surface. The proteins captured by the chip surface are ionized in the ion source and their weight is detected by referring to the different flight time in the flight tube. In this platform, protein chip is the core of the whole system.

Distinguished by chemical modification, the protein chip is divided into hydrophobic surface (H4), normal phase (NP), weak cation exchange (WCX), strong anion exchange (SAX) and immobilized metal affinity capture (IMAC) which are aim to fit for different detecting requirement. Different chemisorption media allows an extremely large amount of proteins to be reduced to a relatively low level, those remained proteins can be absorbed by the surface of chip. They are characterized by: (1) can be directly used for crude biological samples, such as serum, urine, body fluid and cell lysis solution, etc. (2) low dosage of sample is required, generally is 0.5-15l or 2000 cells; (3) high throughput with automatic operation; (4) rapid discovery for multiple biomarkers and some low-abundance, smallmolecular-weight proteins; (5)high sensitivity lower the limitation of detection (LOD) to 1fmol (10-15mol); (6) special identification ability for hydrophobic protein, especially for membrane proteins; (7) a high-efficiency and cost-effective system integrates all the protein separation, purification, identification, detection and data analysis process.

The processing and analysis system produces the results in forms like scanning profiling, bar graph and electrophoresis patterns (simulation gel image) and analyses the difference among two or more groups of results to find out the special mass spectra of identification information. The procedure includes: (1) database building; (2) internal and external information calibration; (3) data processing and analysis.

not.

BioExploer ™.

2. TOF MS

1. Magnetic Beads

used in the clinical research of serum peptidomics.

3. Analysis Software BioExploer™

Serum Peptidomics 365

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

Considering features of the serum polypeptide profiling and defects of the ClinProt system,

On one hand, ClinTOF is capable of detecting the biomarker pattern or biomarker profiling indicating specific diseases in the biological liquid; on the other hand, this technology can identify the candidate for a single biomarker. ClinTOF composes of three parts, including magnetic beads, time-of-flight mass spectrometer (TOF MS) and analysis software

The magnetic beads includes hydrophobic magnetic beads, metal affinity magnetic beads, ion exchange magnetic beads, glycoprotein magnetic beads and immunoaffinity magnetic beads. Further, SPE-C magnetic beads, the reagent dedicated for the serum polypeptide fingerprint diagnosis is available. Presently, magnetic beads have been found in biomedicine fields like immuno-magnetic separation (IMS), cell and cell organelle separation, microorganism detection and nucleic acid hybridization. The whole system is

Time-of-flight mass spectrometer (TOF MS) is used to obtain mass ratio and content of proteins captured by magnetic beads. ClinTOF, the clinical mass spectrometer, has adopted the cutting-edge 60Hz pulsed nitrogen laser which allows the data generated at the fastest speed compared with its counterparts. The zoom optics technology is employed with the laser speckle ranging from 50µm to 200 µm (adjustable), also the greatest adjustable range among like products, so that the size of laser speckles can meet the demands of different samples. The unique gap design in ion source has been utilized, keeping ion sources from contaminations and greatly reducing maintenance frequency. The laser system has been added with the laser energy leveling function, presenting more stable light intensity of the laser and more accurate data. The unique touch screen design has integrated MALDI-TOF

BioExploer™ software is used both in processing genetic data and also protein data. BioExploer™ has combined visualized analysis and multiple mathematical algorithms to build pattern recognition models for MS data classification and forecast, and hunt the disease markers from data. It can perform data visualization, data reduction and data

control system and the PC system, making operations more simple and handy.

**1.2.3 The 3rd generation of serum peptidomics research platform: ClinTOF** 

it is fair to say that ClinTOF is the representative of the latest platform up to now.

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 studies regarding tumor diagnosis with satisfying results.

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 characteristic peaks of the same disease among different researchers.
