Structural Peptide and Protein Analysis

this step is not sufficient to accomplish the identification of the analyte (e.g., for de novo protein and peptide analysis), an additional step has to be performed to obtain the sequence of the purified cleavage products.

5. Peptide ladder sequencing is performed through the generation of C-terminal and N-terminal peptide ladders. For this purpose, carboxypeptidases (e.g., car-boxypeptidase Y) are used. Usually, several different carboxypeptidases have to be applied to cover the whole sequence because most of them exhibit preferences to release certain residues out of the chain. The N-terminal information is obtained similarly through treatment with aminopeptidases or controlled Edman degradation. Analysis of the products is performed on reflectron TOF instruments or with electrospray ionization (ESI) MS. PSD MALDI provides a very sensitive alternative analysis format.

A generic outline to the structural analysis includes the following steps:

Modification Analysis

1. Purification of the targeted protein or peptide out of the sample (tissue, cell culture, etc.) via gel electro-phoretic (e.g., two-dimensional), affinity-based, or gel permeation-based techniques.

2. Mass measurement of the intact protein via MALDI-TOF. At this point of time, databases (such as SWISSPROT, PIR, EMBL, and others) can be used to investigate the identity of the targeted protein.

3. Analysis of disulfide bonds and free suflhydryl groups via chemical reduction and carboxymethylation. The protein is measured prior to and after reduction of disulfide bonds (e.g., using mercaptoethanol) and the subsequent carboxymethylation (e.g., via iodaceta-mide). This treatment provides information on the number of disulfide bonds and free sulfhydryl groups, respectively. In addition, this procedure facilitates the subsequent cleavage step into peptide fragments by eliminating secondary and tertiary structures, thus providing additional cleavage sites.

4. Chemical and or enzymatic cleavage into smaller peptide fragments is accomplished through amino acid-specific cleavage reagents. Common reagents to perform specific cleavage reactions are specific proteases (e.g., trypsin and certain endoproteinases), nonspecific enzymes (e.g., chymotrypsin), and chemical agents (e.g., chlorosuccinimide or cyanogen bromide). The cleavage products are usually fractionated by high-performance liquid chromatography (HPLC)-based techniques prior to MALDI-TOF MS-based analysis. The obtained masses provide a specific signature (fingerprint) that can be used in databases searches. If the information generated in

The analysis of posttranslational modifications (PTMs) of peptides and proteins, such as phosphorylation and glycosylation, is important to elucidate the functions and interactions of peptides and proteins. The initial steps to analyze PTMs are similar to those performed for structural analysis. In fact, following the initial purification step, the material will usually be fractionated for structural and modification analyses.

A straightforward method for the analysis of peptide and protein phosphorylation is to generate short peptide fragments and to compare mass spectra recorded before and after treatment with phosphatases. Residues that are potential phosphorylation sites are: serine, threonine, and thyrosine. Antibodies, specific to the phosphorylated form of the protein, provide a convenient tool for purification.

The analysis of glycosylation can be performed through treatment with residue-specific glycosidases. Similar to phosphoproteins, glycoproteins can be specifically purified via affinity chromatography. The residues that are usually potential glycosylation sites are serine, asparagine, and threonine. To release N-linked carbohydrate chains from glycopeptides, endoglycosidases (mainly peptide N-glycosidase F, PNGase F) are used. In addition, chemical reagents such as trifluoromethanesul-fonic acid can be used. O-linked carbohydrate chains are released via reductive p-elimination (through NaBH4 treatment). Comparative analysis of tryptic digests before and after treatment is used to identify the extent of the glycosilation and to identify the glycosilation sites. The structural characterization of carbohydrate site chains is a complex undertaking. Partially, it can be achieved through mass measurement of the isolated carbohydrate

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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