Mass spectrometry determining protein molecular weight

It is also possible to estimate the molecular weight of a protein by means of gel filtration, or from measurement of its rate of migration through an electrophoresis gel (gel electrophoresis), in comparison with a series of standards of known molecular weight. An absolute and very accurate means of determining protein molecular weights is mass spectrometry. 

For accurate determination of protein molecular weight, mass spectrometry and LC-MS have largely displaced SDS-PAGE. However, SDS-PAGE will still be used where estimates of molecular weight suffice or where MS instrument time is limited. 

Another useful physical property of the crystal is its density, which can be used to determine several useful microscopic properties, including the protein molecular weight, the proteinlwater ratio in the crystal, and the number of protein molecules in each asymmetric unit (defined later). Molecular weights from crystal density are more accurate than those from electrophoresis or most other methods (except mass spectrometry) and are not affected by dissociation or aggregation of protein molecules. The proteinlwater ratio is used to clarify electron-density maps prior to interpretation (Chapter 7). If the unit cell is symmetric (Chapter 4), it can be subdivided into two or more equivalent parts called asymmetric units (the simplest unit cell contains, or in fact is, one asymmetric unit). For interpreting electron-density maps, it is helpful to know the number of protein molecules per asymmetric unit. 

The advent of both ESI and MALDI revolutionized the analysis of large biomolecules of low volatility such as peptides and proteins by their capability to form stable ions with little excess energy, enabling the determination of molecular weights even in protein mixtures. To obtain information specific to the primary structure of proteins, however, principles such as the activation of molecules via collisions with small neutral molecules, which have been used in the study of gaseous ion chemistry for decades, had to be adapted and helped to propel mass spectrometry to being of the most important tools in the field of proteomics. 

Mass spectrometry can determine the molecular weights of peptides and proteins with mass accuracies orders of magnitude better than the molecular weights determined by gel electrophoresis. It is important to note that in determining molecular... 

Outline the application of mass spectrometry to the analysis of proteins and compare the merits of the various methods of determining the molecular weights of proteins. 

An approach that has been developed to determine the molecular weight of large proteins is called the matrix-assisted laser desorption /onizaf/on-time-of-flight (MALDI-TOF) mass spectrometry. Figure 9.11 contains the parent ion peaks for the two model proteins. 

Reversed-phase HPLC is widely utilized to generate a peptide map from digested protein, and the MS online method provides rapid identification of the molecular mass of peptides. The HPLC-MS-FAB online system is a sensitive and precise method for low-MW peptides (<3000 Da) even picomol quantities can be detected. However, as the MW of the analytes increases, the ionization of peptides becomes more difficult and decreases the sensibility of the FAB-MS (112). Electrospray ionization (ESI-MS) was found to be an efficient method for the determination of molecular masses up to 200,000 Da of labile biomolecules, with a precision of better than 0.1%. Molecular weights of peptide standards and an extensive hydrolysate of whey protein were determined by the HPLC-MS-FAB online system and supported by MALDI-TOF (112). Furthermore, HPLC-MS-FAB results were compared with those of Fast Performance Liquid Chro-motography (FPLC) analysis. Mass spectrometry coupled with multidimensional automated chromatography for peptide mapping has also been developed (9f,l 12a). 

The advent of FAB mass spectrometry has allowed the routine molecular weight determination of polar molecules, without derivatization, up to ca 3,000 Daltons, and in exceptional cases, within 1 mass unit to the region of 8,000 Daltons. This advance, coupled with FAB fragmentation, and enzymic digestion techniques, has allowed the rapid solution of a number of problems in protein and peptide chemistry - problems which were hitherto rather difficult to solve. Examples are given. 

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