Peptides/proteins molecular mass determination

Biology MALDI-TOF Peptide mass maps Intact protein molecular mass determination... 

ESI is frequently applied in the various stages of the characterization of peptides and proteins molecular mass determination, amino acid sequencing, determination of nature and position of chemical and posttranslational modifications of proteins, investigation in protein tertiary and quaternary conformations, and the study of noncovalent associates. In most cases, no online separation is applied, but the sample solution is introduced directly via the ESI or... 

The techniques described thus far cope well with samples up to 10 kDa. Molecular mass determinations on peptides can be used to identify modifications occurring after the protein has been assembled according to its DNA code (post-translation), to map a protein structure, or simply to confirm the composition of a peptide. For samples with molecular masses in excess of 10 kDa, the sensitivity of FAB is quite low, and such analyses are far from routine. Two new developments have extended the scope of mass spectrometry even further to the analysis of peptides and proteins of high mass. 

Li, Y. Mclver, R. T., Jr. Hunter, R. L. High-accuracy molecular mass determination for peptides and proteins by Fourier transform mass spectrometry. Anal. Chem. 1994, 66, 2077-2083. 

Figure 8.1) [28]. Thus the molecular mass determined by these techniques corresponds to that calculated using the chemical mass of each element present in the protein. Both values are significantly different from one another. Indeed, the difference between the isotopic mass and the chemical mass of a peptide or of a protein is about 1 Da per 1500 Da. Hence, specifying the mass we are talking about is important. The choice is determined by the resolution of the analyser and the mass of the ion being analysed. 

Mass spectrometry allows the detection and characterization of mutations within proteins, whether they are natural or obtained through directed mutagenesis [97-100]. The approach generally involves three steps molecular mass determination of the intact protein to detect the mutation, PMF of an enzymatic digest to identify mutated peptides, and finally, MS/MS to determine or confirm the position and the nature of the amino acid that has mutated. 

Table 8.6 lists the mass differences due to the substitutions of a given amino acid by another in the sequence of a peptide chain [101]. The accuracy of the molecular mass determination is critical for the success of the mutation characterization. The required accuracy depends not only on the determined molecular mass, but also on the detected substitution. Indeed, the characterization of Gln/Lys substitution needs less accurate mass measurement with a 1 kDa peptide than with a 40kDa protein. In the same way, the characterization of Gly/Trp substitution is easier than the characterization of Gln/Lys substitution. 

The approaches described in the previous section enable the molecular-mass determination of intact proteins, generally with an accuracy better than 0.01%. Further stractural characterization of the protein requires determination of possible post-translational modifications (PTM) as well as the amino acid sequence. In addition, issues related to tertiary and quaternary stracture of the protein, the presence of cofactors, etc., may be relevant. LC-MS plays an important role in the primaiy and secondaiy stractural characterization of proteins, i.e., in terms of amino-acid sequencing and PTM. The procedure generally involves chemical or enzymatic treatment of the intact protein, acquisition of a peptide map or peptide mass fingerprint by either direct infusion (nano-)ESI-MS or RPLC-MS, and the amino-acid sequencing of individual peptides by means of product-ion MS-MS. Further experiments may be needed in relation to PTM, as outlined in more detail in Ch. 19. 

The introduction of soft ionization techniques such as ESI and M ALDI brought tremendous progress in on- and offline characterization of electrophoretically separated peptides and proteins by MS [50]. Combination of CE with MS techniques allows not only high-accuracy molecular mass determination of peptides and proteins separated by CE, but also it provides important structural data on amino acid sequence, the sites of posttranslational modifications, peptide mapping, and the noncovalent interactions of peptides and proteins. 

Extremely rapid RP separations of proteins have been demonstrated on poly(styrene-co-divinylbenzene) monolithic columns. A fast gradient and a flow rate of lOmL/min were used to separate live model proteins in less than 20 s on a 50 X 4.6-mm ID column [53].The same type of monolithic structure has also been prepared in capillary columns of 200 pm ID and showed excellent performance in the identification of proteins by LC-MS through peptide mass fingerprinting and accurate intact molecular mass determination [24]. 

Amino acid analysis and peptide mapping are standard methods in the course of protein identification processes [5, 6]. Molecular mass determination of whole molecules as well as peptide fragments with accuracies of about 0.01% by either matrix-assisted laser desorp-tion/ionization (MALDI)-MS for surface immobilized samples, or electrospray ionization (ESI)-MS for liquid samples, is another highly efficient protein identification method. These methods additionally support the identification of posttranslational modifications such as... 

Peptide Mapping If the sequence of the protein is known, molecular mass determination of the peptide fragments in the unfractionated protein digest can rapidly identify the presence of phosphopeptides. Protein digest is analyzed by MALDI-MS (or ESI-MS), and phosphopeptides are recognized from the mass shift of 80 Da (or a multiple of 80 Da). 

LC-MS finds wide application in the analysis of compounds that are not amenable to GC-MS, i.e. compounds that are highly polar, ionic and thermo-labile, as well as (bio)macromolecules. In environmental applications, LC-MS is applied, often in combination with off-line or on-line solid-phase extraction, to identify pesticides, herbicides, surfactants and other environmental contaminants. LC-MS plays a role in the confirmation of the presence of antibiotic residues in meat, milk and other food products. Furthermore, there is a substantial role for LC-MS in the detection and identification of new compounds in extracts from natural products and the process control of fermentation broths for industrial production of such compounds, e.g. for medicinal use. LC-MS technology is also widely applied in the characterization of peptides and proteins, e.g. rapid molecular-mass determination, peptide mapping, peptide sequencing and the study of protein conformation and noncovalent interactions of drugs, peptides and other compounds with proteins and DNA. However, the most important application area... 

Technologies that offer the opportunity to use MS for intact protein mass determination allow rapid assessment of signal peptide cleavage sites and post-translational heterogeneity. A well-resolved mass spectrum of an intact protein defines its native covalent profile as well as its associated heterogeneity. A comprehensive mass spectrometric approach that integrates both intact protein molecular mass measurement (top-down) and proteolytic fragment identification (bottom-up) would represent a powerful approach for proteome/ protein characterization. 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

In a separate study, a protocol for Matrix-assisted laser desorption-ionization (MALDI) imaging mass spectrometry (IMS) has been proposed.18 This IMS technique provides a new approach to visualize spatial distribution of thousands of molecular species, including peptides, proteins, and their metabolites in two- or three-dimensional levels. This approach may also provide a straightforward method of determining the tissue distribution of multiple peptides or proteins in a quantitative manner.18 Chu et al.19 reported a nondestructive molecular extraction method to obtain proteins from a single FFPE or frozen tissue section, without destroying the tissue morphology, such... 

Relative molecular mass distributions for components of biochemical and polymer systems can be determined with a 10% accuracy using standards. With biochemical materials, where both simple and macro-molecules may be present in an electrolyte solution, desalting is commonly employed to isolate the macromolecules. Inorganic salts and small molecules are eluted well after such materials as peptides, proteins, enzymes and viruses. Desalting is most efficient if gels with relatively small pores are used, the process being more rapid than dialysis. Dilute solutions of macro-molecules can be concentrated and isolated by adding dry gel beads to absorb the solvent and low RMM solutes. 

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