Biomolecule Molecular Weight Determination

There are several computer algorithms, normally incorporated into the instrument software, used to calculate MW. The algorithms are based on the following simple assumptions and calculations. If two adjacent peaks in the spectmm (pi and 

By using two contiguous peaks, the molecular mass of a biopolymer can be calculated by solving Eqs. 15.10 and 15.11. 

TABLE 15.7. Calculated Relationship between Molecular Ion Mass and mlz Values for Multiple Charged (Protonated) Molecular Ions 

Mass (Afr) of Charges Ion Mass mlz between Isotopic Peaks 

Isotope peaks observed for proteins are mainly produced by the 12C and 13C mass difference of 1 mass unit. Once one molecular ion charge state is determined, the other charge states are readily assigned by spectral observation. ESI, MALDI, and FAB are usually used in the positive-ion mode, especially for protein MW determination therefore the proton mass must be used to calculate the ion mass, as shown in Table 15.7. 

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ESI is suitable for almost all kinds of biomolecules, as long as they are polar and soluble in a solvent system that can be used for spraying. Peptides, proteins, carbohydrates, DNA fragments and lipids are all commonly analysed via ESI-MS. Molecular weight determination is one of the main applications. Eurthermore, sequencing of peptides and DNA fragments (section 6.3) is possible with ESI connected to a tandem mass spectrometer (ESI-MS/MS). 

Aurora Biomolecules dedicates to peptide synthesis (and polyclonal antibody production) for any small quantity purpose. FMOC chemistry (on Perceptive Biosystems Pioneer instruments) is used for peptides synthesis Online monitoring of the coupling efficiencies and HATU activation helps insure that the major component of the synthesis is the correct oligopeptide. Purification is firstly carried out by size exclusion chromatography, and then by HPLC on a PE vision purification workstation. Typically, 20 mg of pure peptide are obtained. The molecular weight of the purified peptide is determined as a final confirmation of quality. 

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 molecular weight is an intrinsic property of biomolecules. Sedimentation velocity is one of physicochemical methods which have been employed to determine molecular weights of many biomacromolecules. The molecular weight (M) of a biomacromolecule can be computed according to the Svelberg equation ... 

X-ray diffraction (crystallography) has been around for much longer than NMR and has been used to determine the precise three-dimensional stmcture of biomolecules as large as viruses (molecular weight in the tens of millions ). This technique requires a high-quality crystal and calculates a three-dimensional map of electron density for the molecule. The... 

Size-exclusion chromatography (SEC) is used for the separation of large molecules according to their effective size in solution. In combination with ICP-MS it allows the molecular weight of biomolecules in nondenaturing conditions to be estimated, and it is frequently employed when dealing with complex samples for fractionation purposes, that is, for the identification of various classes of species of an element and to determine the sum of its concentrations in each class. 

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. 

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a classic technique used for the separation and molecular weight (MW) determination of biomolecules. Unfortunately, the technique gives only a rough estimation of MW, with values of 5-10 % being typical. Additionally, it can be subject to systematic errors if the species under investigation has different electrophoretic migration behavior then the MW markers. 

Molecular weights are now readily determined for these molecules with very high precision and accuracy. Variation between the experimental and the predicted MW of a protein (predicted from its DNA code) help in the determination of post-translational modifications of the polypeptide chain. Noncovalent interactions between biomolecules and ligands may also be studied using soft ionization methods. 

Determination of the molecular weight of native pectin presents the usual problems of large, fragile biomolecules, which need only a few chemical events to drastically lower the measured molecular weight, and these are compounded... 

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