**1.1.1 Proteomics**

The performer of life functions is the dynamically-changing protein, rather than the relativelystatic gene. Accordingly, the study of protein is of practical significance in the explication of vital phenomena, especially in the revelation of onset, deterioration and outcome of human diseases, which is also a driving force for the emergence of proteomics. Wilkins and Williams (Wasinger VC. et al, 1995) initiated the study of proteomics by putting forward the concept of proteomics for the first time. The proteome is the set of expressed proteins in a given type of cells, tissues or an organism at a given time under defined conditions. Proteomics is the largescale study of proteomes, to discover composition and expression of proteins in organism, to understand interactions between proteins and explore functions of proteins and laws of vital activities of cells. It covers expression proteomics, functional proteomics and cell-localization proteomics etc. The proteomics technologies has provided a new tool for studying the biomarkers, pathogeny mechanism, diagnostic methods of diseases.

### **1.1.2 Serum proteomics**

When proteomics comes to the clinical applications, it mainly refers to serum proteomics. The features of serum proteome research: firstly, it is easily to access samples, which means that it is able to meet the research requirements and easy to standardize; secondly, the dynamic variation in serum proteins is capable to reflect the pathological changing state of human organs; this is of far-reaching importance for disease diagnosis and curative effect monitoring. Human Proteome Organization (HUPO) brought the human plasma/serum proteome plan under the first-phase of the human proteome plans, which is showing the significance of studying serum proteomics for healthy and sick states of human.

Taking all proteins expressed in the serum of selected target clusters as the object, the serum proteomics, based on the normal protein expression profiles, aims to look for the differential proteins and define disease-associated proteins, the structures and functions of which will further be studied. In the hope of presenting a new approach for studying the pathological and physiological mechanisms of severe diseases, specific protein markers are expected to

Serum Peptidomics 363

Such are three major technical approaches for proteomics study as two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) and bioinformatics. Similar to the proteomics technology, serum peptidomics technology also involves in technical advancement and upgrade in sample preparation, detection and result analysis, which jointly constitute a platform in serum peptidomics research. Most of studies rely on matrixassisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The working principle of MALDI-TOF-MS is: the pulse laser is utilized to force the matrix to absorb energy from laser so that the solid-phase polypeptide samples are ionized; the ionized peptides are put into the TOF mass analyzer and separated due to difference in the mass-to-charge ratio (m/z); the peptide mass fingerprint (PMF), peptide sequence tag (PST) or the amino acid sequences can be obtained through measuring peptide ions; qualitative identification or quantitative analysis of peptides can be accomplished through searching in the proteome database with corresponding software. On this basis, three serum peptidomics

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

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

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

separation, purification, identification, detection and data analysis process.

information calibration; (3) data processing and analysis.

**1.2 History of serum peptidomics platform** 

research platforms have been developed successfully.

core of the whole system.

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

be found for early diagnosis and drug targets. Compared with the tissue proteins and cell proteins, serum proteins are unique in many aspects such as the largest number (tens of thousands of varieties), extremely great difference in contents of proteins (with a difference of more than 108-1012), extremely instable variety and content of low-abundance proteins. The main components of serum proteins are albumin and immunoglobulin, which share the features like high abundance, large molecular weight and easy-to-detect. Besides, nonprotein substances like lipids and salts also exist in the serum. All other components will interfere with the study of small-molecular-weight proteins in the serum. Usually, all the important information come from the low-abundance proteins and small-molecular-weight proteins with great varieties and different properties, on the other hand, the features of these proteins make them difficult to be separated and identified.

#### **1.1.3 Serum peptidomics**

With the deepening of biological study of proteins, a kind of non-protein intermediate that composes of amino acid is discovered and described as polypeptide. According to biochemists, peptides are short polymers of amino acids linked by amino bonds (also called peptide bonds). Polypeptide is a kind of peptides with more than 10 amino acids (if fewer than 10, they are called oligo-peptides). In our study here, proteins with the molecular weight less than 10KDa fall into the category of the polypeptides. Besides molecular weights, the polypeptides are also different from proteins in functions: firstly, the polypeptide are information messenger, which can arouse various physiological activities and regulate biochemical response; secondly, polypeptide have high bioactivity; thirdly, as smaller molecule, the structure of polypeptide is easier to rebuild and simpler for artificial synthesis and chemosynthesis; fourthly, fragments of polypeptide can be used for further research of protein features and changing and synthesizing proteins as basic materials. The polypeptide is a sort of biologically active substance related to cell functions of organisms. Tens of thousands of polypeptides have been found existing in organisms and can be synthesized in all cells. Moreover, nearly all cells are regulated by polypeptides, which play a role in hormone, nerves, cell growth and reproduction, etc. Presently, in company with the instant development of proteomics and mass-spectrometric technology, more and more researchers are turning their eyes to polypeptide, which thereby bring about the proteomicsbased peptidomics.

The main clinical application of peptidomics is the serum peptidomics. The serum peptidome, generally referring to serum peptidome profiling, is to detect the accurate mass value of polypeptide in serum by mass spectrometry and process mass spectrum with the bioinformatics method to build a polypeptide profiling. In the profiling, the peptidome identified by an accurate mass value can be further analyzed into amino acid sequence through tandem mass spectrometer, thereby used to identify the precursor proteins and their biogenetic derivation. Through contrasting differences in the serum peptidome profiling of the patients and the healthy controls, proteins or polypeptides specifically expressed in disease state and the biomarkers associated with diseases can be discovered to perform the studies of proteins related to early diagnosis, classification and subtype, onset mechanism of diseases. The serum polypeptide profiling is reputed to be a "brand-new health fingerprinting library" technology (Tarnaris A et al, 2006) and has become a research hotspot of proteomics.

be found for early diagnosis and drug targets. Compared with the tissue proteins and cell proteins, serum proteins are unique in many aspects such as the largest number (tens of thousands of varieties), extremely great difference in contents of proteins (with a difference of more than 108-1012), extremely instable variety and content of low-abundance proteins. The main components of serum proteins are albumin and immunoglobulin, which share the features like high abundance, large molecular weight and easy-to-detect. Besides, nonprotein substances like lipids and salts also exist in the serum. All other components will interfere with the study of small-molecular-weight proteins in the serum. Usually, all the important information come from the low-abundance proteins and small-molecular-weight proteins with great varieties and different properties, on the other hand, the features of

With the deepening of biological study of proteins, a kind of non-protein intermediate that composes of amino acid is discovered and described as polypeptide. According to biochemists, peptides are short polymers of amino acids linked by amino bonds (also called peptide bonds). Polypeptide is a kind of peptides with more than 10 amino acids (if fewer than 10, they are called oligo-peptides). In our study here, proteins with the molecular weight less than 10KDa fall into the category of the polypeptides. Besides molecular weights, the polypeptides are also different from proteins in functions: firstly, the polypeptide are information messenger, which can arouse various physiological activities and regulate biochemical response; secondly, polypeptide have high bioactivity; thirdly, as smaller molecule, the structure of polypeptide is easier to rebuild and simpler for artificial synthesis and chemosynthesis; fourthly, fragments of polypeptide can be used for further research of protein features and changing and synthesizing proteins as basic materials. The polypeptide is a sort of biologically active substance related to cell functions of organisms. Tens of thousands of polypeptides have been found existing in organisms and can be synthesized in all cells. Moreover, nearly all cells are regulated by polypeptides, which play a role in hormone, nerves, cell growth and reproduction, etc. Presently, in company with the instant development of proteomics and mass-spectrometric technology, more and more researchers are turning their eyes to polypeptide, which thereby bring about the proteomics-

The main clinical application of peptidomics is the serum peptidomics. The serum peptidome, generally referring to serum peptidome profiling, is to detect the accurate mass value of polypeptide in serum by mass spectrometry and process mass spectrum with the bioinformatics method to build a polypeptide profiling. In the profiling, the peptidome identified by an accurate mass value can be further analyzed into amino acid sequence through tandem mass spectrometer, thereby used to identify the precursor proteins and their biogenetic derivation. Through contrasting differences in the serum peptidome profiling of the patients and the healthy controls, proteins or polypeptides specifically expressed in disease state and the biomarkers associated with diseases can be discovered to perform the studies of proteins related to early diagnosis, classification and subtype, onset mechanism of diseases. The serum polypeptide profiling is reputed to be a "brand-new health fingerprinting library" technology (Tarnaris A et al, 2006) and has become a research

these proteins make them difficult to be separated and identified.

**1.1.3 Serum peptidomics** 

based peptidomics.

hotspot of proteomics.
