生物化学技术

Arachidonic acid located at the sn-2 position of glycerophospholipids is a precursor of an important class of lipid mediators termed eicosanoids (1,2). The major eicosanoids include leukotrienes, prostaglandins, and thromboxanes. In inflammatory cells, the bulk of AA is esterified ...

Western blotting provides an effective, sensitive, and specific method for the identification and quantitation of cyclooxygenase protein expression in an unknown complex protein sample. Electrophoresis is used to separate complex mixtures of proteins. Denaturing discont ...

The release of arachidonic acid (AA) from membrane phospholipids by phospholipase A2 (PLA2) is a critical step in the generation of lipid mediators by mast cells. AA released by PLA2 activity is metabolized to bioactive compounds, such as prostaglandins, thromboxane, and leukotrienes. D ...

An important goal in proteomics is to compare the relative amounts of different proteins in biological samples and to try to correlate these differences with changes in physiological state. The isotope- coded affinity tag technique pioneered in Aebersold’s laboratory takes advanta ...

Protein glycosylation has long been recognized as a very common posttranslational modifi- cation. Protein glycosylation is prevalent in proteins destined for extracellular environments. These include proteins localized on the extracellular surface and those secreted to b ...

Multidimensional chromatography coupled to mass spectrometry is an emerging technique for the analysis of complex protein mixtures. One approach in this general category, multidimensional protein identification technology (MudPIT), couples biphasic or triphasic micr ...

N-glycosylation of proteins is the predominant glycosylation in mammals and confers specific conformations, localization, and functions to proteins. High-throughput proteomics techniques have focused on the identification of proteins through amino acid sequence dete ...

This chapter describes the use of an open-source, freely available informatics system for the identification of proteins using tandem mass spectra of peptides derived from an enzymatic digest of a mixture of mature proteins. The chapter describes the use of features of the Global Proteome M ...

In this chapter, we address the issue of matrix-assisted laser desorption/ionization mass spectrometry (MS) data analysis for disease biomarker discovery. We first give a general framework of MS data analysis, then focus on several key steps. After that, we show some application examples u ...

Protein microarrays are a miniaturized format for displaying in close spatial density hundreds or thousands of purified proteins that provide a powerful platform for the high-throughput assay of protein function. The traditional method of producing them requires the high-throu ...

Proteolysis is a key mechanism for protein homeostasis in living cells. This process is effected by different classes of proteases. The proteasome is one of the most abundant and versatile proteases, bearing three different proteolytic active sites. The proteasome plays an important ro ...

Antibody microarray measurements show great potential for the simultaneous quantification of many proteins in small amounts of body fluids and extracts. Over the last few years, a microarray platform centered around the concept of microarrays in microtiter wells was developed, and f ...

There are many instances in which it is desirable to generate profiles of the relative abundance of a multiplicity of protein species. Examples include studies in embryonic development, immunobiology, drug responses, cancer biology, biomarkers, and so on. Microarray formats provide a ...

Size-exclusion chromatography (SEC), coupled with “on-line” static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection, provides a universal approach for determination of the molar mass and oligomeric state in solution of native proteins as well as gly ...

The combination of surface plasmon pesonance (SPR) and mass spectrometry (MS) provides a unique methodology for studying proteins and their interactions. SPR is utilized to assess protein quantitative variations and the kinetic aspects of protein interactions, whereas MS comple ...

The surface-sensitive optical technique of surface plasmon resonance (SPR) imaging is an ideal method for the study of affinity binding interactions of unlabeled biological molecules in a multiplexed format. This approach has been widely applied to monitor DNA-DNA, DNA-RNA, peptide ...

Affinity mass spectrometry (AMS) is a proteomics approach for selectively isolating target protein( s) from complex biological fluids for mass spectrometric analysis. The resulting high-content mass spectrometry (MS) data show the unique MS protein signatures (wild-type, post ...

As the field of clinical proteomics progresses, discovery of disease biomarkers becomes paramount. However, the immediate challenges are to establish standard operating procedures for both clinical specimen handling and reduction of sample complexity and to increase the abil ...

Successful collection of tissue samples for molecular analysis requires critical considerations. We describe here our procedure for tissue specimen collection for proteomic purposes with emphasis on the most important steps, including timing issues and the procedures for imm ...

Preanalytical variables can alter the analysis of blood-derived samples. Prior to the analysis of a blood sample, multiple steps are necessary to generate the desired specimen. The choice of blood specimens, its collection, handling, processing, and storage are important aspects since ...