Adapted from Hoehn and Suffredini.12
笔记详情
标题
蛋白质组学的分析工具一览表
内容
Analytical Tools in Proteomics
| Method | Principle | Advantages | Disadvantages | |
|---|---|---|---|---|
| Electrophoresis | ||||
| Electrophoresis | When an electric field is applied to a solution containing a protein that has a net positive or negative charge, the protein migrates at a rate that depends on its net charge, size, and shape. | Gels must be stained before proteins can be visualized. Rarely useful by itself as proteins cannot be accurately identified without the use of another detection technique such as immunoblotting or mass spectroscopy. | ||
| SDS-PAGE | Proteins migrate through inert matrix gel of polyacrylamide. Pore size is adjustable to retard protein of interest. SDS is a negatively charged detergent that unfolds proteins and frees them from other molecules. Proteins migrate at different rates toward positive electrode. | Separates all types of proteins, even those insoluble in water. | One-dimensional separation method has limited resolution. Closely spaced bands or peaks tend to overlap. Can only resolve a small number of proteins. | |
| Two-dimensional gel electrophoresis | Combines 2 separation procedures. First dimension: the solubilized, denatured proteins are separated by their isoelectric point (pH where net charge is 0) in a polyacrylamide gel. Second dimension: the narrow gel containing proteins separated by isoelectric focusing undergoes electrophoresis at a right angle in SDS-PAGE to separate by size. | Good resolution of mixture. Comparison of multiple gels facilitated by image analysis software. Posttranslational modifications can be discerned. Resolution of protein approximately 1 ng/mL. | Presence of high abundance proteins (i.e., albumin, immunoglobulins) may obscure low abundance proteins. Low throughput. Final identification of protein requires spot removal from gel, digestion, and analysis of peptides by mass spectrometry. Unable to resolve low molecular weight proteins (<10,000 Da). Not easily amenable to multivariate analysis. | |
| Two-dimensional fluorescence difference gel electrophoresis | Labels complex mixtures with fluorescent dyes before conventional two-dimensional electrophoresis. Different cyanine dyes are used to label protein from different samples and will be excited and emit at different light wavelengths. Up to three different samples can be labeled and mixed together (test, control, reference). | Analysis of differences between mixtures is simplified. Ratio of protein expression can be obtained in a single gel, and an internal standard can be used in each gel to reduce gel-to-gel variation. Very sensitive. | Presence of high abundance proteins (i.e., albumin, immunoglobulins) may obscure low abundance proteins. Low throughput. Final identification of protein requires spot removal from gel, digestion, and analysis of peptides by mass spectrometry. Many spots cannot be identified because of lack of material. Unable to resolve low molecular weight proteins (<10,000 Da). | |
| Protein array | ||||
| Protein arrays | Multiplex protein arrays, cytokine arrays, tissue microarrays | In most common form, antibodies to known proteins are tethered to a surface (beads, nitrocellulose, etc.) and then detected using principles of immunoassays. | High sensitivity and throughput. Multiple analytes can be measured simultaneously. Identification of potential targets already known. | Limited antibody availability and specificity. Required some prior knowledge of expressed proteins. May not detect isoforms of analyte. Cost per sample may be prohibitive. |
| Mass spectroscopy (MS) | ||||
| MS | Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) | Determines the precise mass of protein or peptide fragment from protein. Protein/peptide samples are mixed with organic acid matrix, dried on metal slide, and blasted by laser ionizing the peptide, which is accelerated in an electric field toward a detector. The time it takes to reach the detector is determined by the charge and mass. Peptide sequence information can be obtained with tandem mass spectrometers (MS–MS). | Highest resolution is for molecules <3,000 Da in size. | |
| Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) | Comparable to MALDI, the difference being that SELDI uses chromatographic chip arrays to selectively bind subsets of proteins from complex samples. The surfaces can be washed to remove nonspecifically bound proteins and substances that can interfere with the ionization process (salt, detergents, etc.). | High throughput via automation. Requires minimal sample preparation. Can be combined with prefractionation of material to enhance the detection of lower abundant proteins. | No direct identification of proteins. Less sensitive to high molecular weight protein (>20 kDa). May have instrument-to-instrument variation. | |
| Stable isotope labeling | Biological samples are labeled with different stable isotopes using modifying agents targeting a specific amino acid (e.g., ICAT). After separation and mass spectrometry, peptides from the 2 samples differing in mass units specific for the isotope used (e.g., 8-Da mass shift for ICAT) can then be used to provide relative quantification. | Wider proteome coverage than other methods. Can obtain quantitative information on a large number of proteins; Usually yields IDs of relevant proteins. | Technically demanding; very low throughput capability; samples need to be trypsinized before analysis; reliable quantitative measurements likely on most abundant proteins. | |
来源
j gen intern med. 2008 january 23(suppl 1): 78–84.
类别
领域
cancer/oncology
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