Molecular mass, determination

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. 

The molecular mass determined osmometrically corresponds to the formula S5O. The SO stretching vibration was observed in the infrared spectrum at 1119 cm (at -65 °G) indicating an exocyclic sulfoxide group similar to the one in SsO (see below). At -50 °G the solution of S5O may be kept for several days without decomposition which usually results in a Tyndall effect caused by a colloidal polymeric sulfuroxide which is the expected decomposition product. At 25 °G some decomposition already occurs within... 

The third described enzyme form with pH optimum about 4.7 [11, 4], we found in Fraction C - the fraction from carrot roots pulp (Fig. 2). We supposed that this form of exopolygalacturonase is relatively strongly bound on carrot cell walls and so it can be released only by higher salt concentrations. The approximative molecular mass determination on Superose 12 (Fig. 3c) showed the molecular mass about 50 000 for this form and the second, with more acidic pH optimum, form present in the fraction. The further characterization of these enzymes showed the exopolygalacturonase with pH optimum 4.7 to be identical with enzyme described sooner by Pressey and Avants [4] and exopolygalacturonase with pH optimum 3.8 to be identical with the enzyme from Fraction A. In conclusion, the exopolygalacturonase form with pH optimum 3.8 can be considered to be the main enzyme form present in carrot roots. 

Fig. 5. The approximate molecular mass determination of polygalacturonase [(0—0) - substrate 0.5% pectate, pH 4.6] and exopolygalacturonase [( — ) - substrate 1.0 pmol/ml of di(D-galactosiduronic) acid, pH 4.0] on Superose 12 column (FPLC device). Flow rate 0.5 ml/min. System 0.05 M phosphate buffer pH 7.0, 0.15 M NaCl. Standarts Ferritin (450 kDa), Katalase (240 kDa), Aldolase (158 kDa), Albumin (68 kDa), Albumin (45 kDa), Chymotrypsinogen A (25 kDa), Cytochrome C (12.5 kDa).

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. 

End group quantification is thus a useful method for number average molecular mass determination. Also, if the end group concentration is higher than the value calculated from an independent Mn determination, chain branching can be deduced and quantified. 

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. 

The reported amino acid composition cannot be correct. The minimum molecular mass calculated from it is about 120 u higher than the molecular mass determined by mass spectrometry. Also the amino acids acting as ligands for Fe " are missing 2D-ser, 2L-Ser... 

Meyer JM, Gruffaz C, Raharinosy V, Bezverbnaya I, Schafer M, Budzikiewicz, H (2008) Siderotyping of Fluorescent Pseudomonas Molecular Mass Determination by Mass Spectrometry as a Powerful Pyoveidine Siderotyping Method. Biometals 21 259... 

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. 

Molecular exclusion chromatography is based on the inability of large molecules to enter small pores in the stationary phase. Small molecules enter these pores and therefore exhibit longer elution times than large molecules. Molecular exclusion is used for separations based on size and for molecular mass determinations of macromolecules. In affinity chromatography, the stationary phase retains one particular solute in a complex mixture. After all other components have been eluted, the desired species is liberated by a change in conditions. 

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... 

Raman spectra. In a collision between a photon and a molecule, the photon may undergo elastic collision in which the photon loses no energy but changes its direction of travel. Such scattering is known as Rayleigh scattering and forms the basis for a method of molecular mass determination. Sometimes inelastic collisions occur in which both the... 

Aluminum, gallium, indium, and thallium have been successfully incorporated in cyclic systems, although little structural information has been obtained (75). Hoberg (34) has prepared many such ring compounds, including, for example, 8a and 8b, and has demonstrated the former to be dimers from molecular mass determinations and from mass spectra. 

We said in the previous section that elemental analysis gives only an empirical formula. To determine a compound s molecular formula, it s also necessary to know the compound s molecular mass. How is molecular mass determined ... 

The molecular mass of ethylene glycol (Problem 3.109) is 62.0689 amu when calculated using the atomic masses found in a standard periodic table, yet the molecular mass determined experimentally by high-resolution mass spectrometry is 62.0368 amu. Explain the discrepancy. 

Which of the four colligative properties is most often used for molecular mass determination, and why ... 

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

Modern mass spectrometers make it possible for substances in the femtomole range (10-15M) to be analyzed, and for high molecular masses of complex molecules well over 100 kDa to be obtained. Mass spectrometers allow molecular mass determination, and the generation of fragmentation data to allow sequencing and structure elucidation, generally in tandem mass-spectrometric experiments. 

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

In view of experimental simplicity and accuracy, viscosity measurements are extremely useful for routine relative molecular mass determinations on a particular polymer-solvent system. K and a for the system are determined by measuring the intrinsic viscosities of polymer fractions for which the relative molecular masses have been determined independently - e.g. by osmotic pressure, sedimentation or light scattering. 

Molecular mass determined from amino acid composition deduced from DNA sequence... 

Molecular mass determined from SDS-PAGE or gelfiltration. 

The absorbance of la remained almost constant even after 850 cycles, while the absorbance of 3a gradually declined. At the same time, a photostable, violet product with an absorption maximum at 547 nm was formed. The photostable by-product could be isolated by HPLC, and was found by elemental analysis and molecular mass determination to be isomeric with compound 3a. Its molecular structure was determined by X-ray crystallographic analysis to be a six-membered condensed ring structure 9, as shown in Figure 3. The by-product was produced from the closed-ring form more efficiently by UV irradiation. 

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