Protein molecular mass determination

Biology TOF-MS (ESI) 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. 

Size-based analysis of SDS-protein complexes in polyacrylamide gels (SDS-PAGE) is the most common type of slab gel electrophoresis for the characterization of polypeptides, and SDS-PAGE is one of the most commonly used methods for the determination of protein molecular masses.117 The uses for size-based techniques include purity determination, molecular size estimation, and identification of posttranslational modifications.118119 Some native protein studies also benefit from size-based separation, e.g., detection of physically interacting oligomers. 

The relative molecular mass determination of an unknown protein is generally performed automatically using various deconvolution algorithms, but the procedure is limited to relatively simple mixtures. 

Masses computed with Equation 22-8 appear in the last column of Table 22-3. A limitation on the accuracy of molecular mass determination is the accuracy of the mlz scale of the mass spectrum. Proteins of known mass can be used to calibrate the instrument. 

Several newer methods of molecular mass determination were developed in the 1960s-1980s. One is gel filtration. A column of gel beads such as Sephadex is prepared carefully and is calibrated by passing a series of protein solutions through it. The volume V, at which a protein peak emerges from the column can be expressed as the sum of two terms (Eq. 3-19) in... 

The product of the crystal density and the unit-cell volume (determined from crystallographic analysis, Chapter 4) gives the total mass within the unit cell. This quantity, expressed in daltons, is the sum of all atomic masses in one unit cell. If the protein molecular mass and the number of protein molecules per unit cell are known, then the remainder of the cell can be assumed to be water, thus establishing the proteinlwater ratio. 

Molecular Mass Determination of Protein Monomer SDS-PACE... 

At low compressions, up to c. 1 mN m i, protein films tend to be gaseous, thus permitting relative molecular mass determination. 

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 membrane-bound H. saccharovorum ATPase can be purified to a specific activity of 3.6(imol Pj/min/mg protein [46]. The native enzyme (Mr 350000) is composed of four subunits. Molecular-mass determinations in the presence of sodium dodecylsulfate (SDS) provide Mr estimates of 87 000, 60 000, 29 000, and 20 000 for the subunits, designated I, II, III, and ly respectively [23], SDS gel electrophoresis overestimates the molecular masses of acidic proteins [70] and more accurate values can be obtained by electrophoresis in the presence of cetyltrimethylammonium bromide [71]. When this is done, the resulting values for Mr agree with the those obtained using SDS [46]. 

A major breakthrough was achieved by Fenn et al. [5-6] using ESI-MS in 1988. They demonstrated the generation of multiple-charge ions from large proteins by ESI-MS, enabling their detection and molecular-mass determination with a relatively inexpensive quadrupole mass analyser. Some of their early mass spectra are shown in Figure 16.2. 

Figure 16.3 Principle of molecular-mass determination from the ion envelope of multiple-charge protein ions.

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. 

A second approach exploits the information contained in genome and protein sequencing databases. The masses of a set of proteins from the bacterial strain under study are determined and used to search databases for protein molecular mass matching. This has been used as a method to differentiate B. subtilis and E. coli, two organisms that have completely sequenced genomes.34... 

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