The human proteome contains more functional polypeptides than all genes contained in the genome, in part due to simultaneous and post-translational protein modifications. The goal of proteomics research is to obtain a complete picture of the functional proteins present in a particular cell or tissue type, and in healthy or diseased tissues. One of the important areas of proteomics research is the identification of post-translationally modified proteins, their modification sites, the function of modifications, and the interaction of modified proteins in cellular functional networks.
In the past few decades, various methods have been developed for the determination of protein modifications. Here, we highlight the general methods for identifying protein modifications, mass spectrometry; specific methods for identifying phosphorylation, phosphate labeling; methods for identifying ubiquitination; and recognition of histone acetylation and methylation during chromatin remodeling and a method for identifying protein glycosylation.
For the past 20 years, mass spectrometry has become an indispensable tool for determining the type and location of protein modifications. Mass spectrometry can be used for purified proteins or mixtures of proteins, such as cell lysates.
The mass spectrometer produces gas phase ions from a protein sample, separates them according to mass to charge ratio (m/z), and records their abundance. Mass spectrometry can be used for molecular weight determination of polypeptides and proteins, determination of polypeptide amino acid sequences, and detection of post-translational modifications, as well as relative quantification of polypeptides and proteins. This method cannot be used for absolute quantification.
Sample preparation by mass spectrometry, is digested into small molecule polypeptide fragments with restriction endonucleases such as trypsin or lysate. These polypeptide fragments were then evaporated and analyzed to determine their m/z values. Since the cleavage sites are known, a specific amino acid sequence for each peptide can be determined by quality using a computer program. Since the molecular weight and charge of a molecule such as a phosphate group are known, phosphorylation of a specific amino acid in a peptide can also be detected.
A matrix-assisted laser desorption/ionization (MALDI) peptide map or nanoliter mass spectrometer can analyze the digested protein sample. However, the limitation of these methods is that all peptide fragments cannot be completely detected, some peptides are clearly visible, and some peptides are not visible. This is often a major problem for the analysis of complex mixtures. For the analysis of modified peptides and post-translational modifications, the peptides are first separated by reverse phase chromatography, then fractions are collected and analyzed by mass spectrometry (LC/MS). In order to more accurately determine the nature of peptide modification, tandem mass spectrometry (MS/MS) experiments are often used. After the first MS step, the peptide ions are struck with an inert gas, resulting in further fragmentation. These polypeptide fragments are then analyzed in a second MS step. During this process, some of the modified peptides will remain unchanged, and the resulting peptide pattern will be similar to the uninterrupted peptides. Some peptides will be significantly fragmented, and the resulting peptide pattern can be the nature and position of the modified amino acids points provide further information.
In addition to being used to determine the modified state of a single protein, mass spectrometry was also used to determine a broad data set of all proteins with a specific modification such as phosphorylation. This usually involves affinity chromatography prior to mass spectrometry. This technique is used to identify “secondary proteome”; with such modifications, phosphorylation proteome analysis of the activation state of the entire signaling network in cancer can be used.
Mass spectrometry has been used to determine all four modifications described above. However, mass spectrometry is expensive, requires specific equipment, and often requires more quantitative data, so mass spectrometry is often used in conjunction with other biochemical methods to analyze post-translational modifications of proteins.
Phosphorylation, or the addition of a phosphate group to a serine, threonine or tyrosine residue, is one of the most common forms of protein modification. In the signal transduction pathway in cells, protein phosphorylation plays an important role and is a reversible, fine-tuned signal. Several in vivo and in vitro methods are used to detect the phosphorylation status of a protein, to detect phosphorylation of a particular amino acid, and to determine whether a particular kinase (or phosphatase) acts on the target protein.
In vitro phosphorylation analysis
The radiometric kinase reaction uses 32P-gamma-ATP and can be used to detect phosphorylation status in vitro. This is also the gold standard for detecting the effects of specific kinases. Purified target protein, kinase, and 32P-gamma-ATP were incubated in reaction buffer. Then, the reaction solution was filtered through a filter, the protein was bound to a filter, the unbound ATP was washed away, and the phosphorylation level (the radioisotope remaining in the filter) was detected by a scintillation counter. Alternatively, the reaction solution was analyzed by SDS-PAGE electrophoresis and observed by X-ray film exposure. This method is semi-quantitative, but the molecular weight of the phosphorylated protein can be determined.
Likewise, the reaction can include a phosphatase, rather than a kinase, to determine if the phosphorylated protein is a substrate for a particular phosphatase.
Radioactive pulse marking
To detect phosphorylation in vivo, radioactive pulse labeling can be used. The cells are grown in the presence of 32P-orthophosphate. A certain antibody is then immunoprecipitated with a specific antibody, and the resulting radioisotope is precipitated by scintillation counter or SDS-PAGE electrophoresis and X-ray film exposure. This method can detect phosphorylation under various physiological conditions. In addition, in combination with protein knockout assays, this method can also be used to analyze whether a particular kinase or phosphatase affects the modified state of the target protein.
There are two classes of phosphorylation-specific antibodies. The first class is the universal phosphorylated tyrosine, phosphorylated serine, phosphorylated threonine antibody, which binds to any phosphorylated tyrosine, phosphorylated serine and phosphorylated threonine molecules, and is not related to adjacent amino acid residues. The second class includes antibodies that phosphorylate specific amino acid epitope antibodies.
After the (hypothetical) phosphorylation site is identified by computer or by mass spectrometry, a universal antibody against the phosphorylation site can be purchased from the company by immunoprecipitation to determine if the target protein is phosphorylated at a particular site (eg, a typical cyclin/cdk site). Antibodies prepared for a particular phosphorylated amino acid, such as phosphotyrosine, can also be used. Next, phosphorylated antibodies were prepared against specific epitopes and phosphorylation was detected by simple immunoblotting. In order to analyze a large number of samples, an enzyme-linked immunosorbent assay can be used to bind the target protein to the membrane and then detect it with a phosphorylated specific antibody.
To be continued in Part Two…