Molecular mass measurement of proteins

The protein spots are picked up with an automatic spot picker for digestion and subsequent mass spectrometry analysis. Alternatively, the separated protein spots are transferred to an inert support (Western blotting). Commonly used inert supports are polyvinylidene difluoride (PVDF) and nitrocellulose. This process cleans and concentrates the separated protein before its further analysis. 

The mass measurement of an intact protein is the first important step in the characterization of that protein. It provides a frame within which the final structure must fit. The molecular mass information can be used for the following applications  

Traditionally, the molecular mass of proteins has been estimated with SDS-PAGE, in which the migration pattern of a protein is compared to a set of known mass proteins. The accuracy of this procedure is, however, very poor (5 to 20%) and mitigates against its use in the applications pointed out above. More accurate mass values are required to distinguish a mutation between Asp and Asn, Asn and lle/Leu, Glu and Gin, and Lys and Glu/Gln. Currently, ESI and MALDl, combined with Q-TOF, orbitrap, and FT-ICR, have become a standard protocol to determine the molecular mass of intact proteins with an accuracy within 0.01%. 

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Another type of ion is formed almost uniquely by the electrospray inlet/ion source which makes this technique so valuable for examining substances such as proteins that have large relative molecular mass. Measurement of m/z ratios usually gives a direct measure of mass for most mass spectrometry because z = 1 and so m/z = m/1 = m. Values of z greater than one are unusual. However, for electrospray, values of z greater than one (often much greater), are quite coimnonplace. For example, instead of the [M + H]+ ions common in simple Cl, ions in electrospray can be [M + n-H]- where n can be anything from 1 to about 30. 

Reduction-alkylation of proteins. The molecular mass measurement of the target protein before and after this step provides information on the number of cysteine residues and disulfide bonds. 

Top-down proteomics. This strategy deals with intact protein molecules no proteolytic cleavage is performed [41], It involves the accurate molecular mass measurement of the intact protein nsing high-resolution mass spectrometry within 2 Da, followed by a molecular mass database search. The identity of... 

Comparative Peptide Mapping This procedure is used when the protein is cleaved with specific cleaving agents and the sequence of the protein is known. A simple molecular mass measurement of the peptides in a protein digest will identify the position of a disulfide bond in the protein. 

Example ESI on a magnetic sector instrument set to R = 20,000 allows for the flail resolution of isotopic peaks in case of medium-molecular weight proteins (Fig. 11.21). This enables the direct determination of the charge state of the ions from the spacing of the isotopic peaks, i.e., 1 1h- for the lysozyme ion due to the average spaces of Am = 0.091 u and 13h- for the myoglobin ion due to Am = 0.077 u. In this particular case, the lysozyme [M+llH]" ion serves as a mass reference for the accurate mass measurement of the unknown" [M+13H] ion. [103]... 

Chaif, B. T., and S. B. H. Kent, Weighing naked proteins Practical, high-accuracy mass measurement of peptides and proteins. Science 257 1885-1893, 1992. Proteins with molecular masses of as much as 100 kd or more can be analyzed at picomole sensitivities to give simple mass spectra corresponding to the intact molecule. This development has allowed unprecedented accuracy in the determination of protein molecular weights. 

Intact protein mass spectrometry allows the molecular mass determination of either proteins or complexes of proteins and covalently bound ligands/other proteins. In a first step, the sample is desalted to detach from buffer components and small ions that would interfere through noncovalent complexes in the gas phase. Next, the isolated protein is ionized, for example, by electrospray ionization (ESI). The acid in the eluent causes protonation of the protein at basic sites, particularly lysine and arginine residues, so that m/z values of multiple species with different charges can be measured in a mass spectrometer. These data are then combined during the deconvolution process to yield the mass of the protein or complex. 

Although of great value, accurate molecular mass measurement is only part of the protein characterization process. In order to confirm a protein sequence the material can be digested to produce peptides that can in turn be characterized by MALDI... 

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