Molecular mass analysis

Figure 13 Molecular-mass analysis of phosphorylase B (panel B) and the separation of the noncovalently (Sypro Red) labeled protein markers (panel A ALA, a-lactalbumin CBA, carbonic anhydrase OVA, ovalbumin BSA, bovine serum albumin BGA, (f-galactosidase) and the covalently (FITC) labeled protein markers (panel C TRI, tripsin inhibitor CAH, carbonic anhydrase ADH, alcohol dehydrogenase BSA, bovine serum albumin BGA, P-galactosidase). Separation conditions gel, 1% agarose, 2% linear polyacrylamide (LPA, MW 700,000-1,000,000) in 50 mM Tris, 50 mM TAPS, 0.05% SDS (pH 8.4) separation buffer, 50 mM Tris, 50 mM TAPS, 0.05% SDS (pH 8.4) separation voltage, 420 V, current, 5 mA gel thickness, 190 pm effective separation length, 3.5 cm temperature, 25°C sample loading, 0.2 pL into 2.5 x 4 x 0.19-mm injection wells. Sample buffer contained 0.05% SDS and 1 x Sypro Red. (Reproduced with permission from Ref. 141.)...

High-resolution molecular mass analysis provides the molecular formula CnH2iO of acanthifolin, which corresponds to seven double bond equivalents. The proton broadband decoupled NMR spectrum (Fig. 6a) displays all 17 carbon atoms of the molecule, including a carboxy group (5c = 170.5) and four additional C atoms in the sp range of chemical shifts (5c = 145.8, 138.1, 119.8 and 116.2), indicating two CC double bonds. Only three of all seven double bonds of the molecule are detected by NMR. To conclude, acanthifolin incorporates a tetracyclic ring system... 

The accuracy of kp determination by PLP-SEC is essentially determined by the quality of molecular mass analysis. In particular, the position of inflection points needs to be precisely... 

As a consequence, for accurate molecular mass analysis of intact proteins, ESI in combination with an orthogonal TOE, electrostatic ion trap or FT-ICR MS is the preferred instrumentation if the samples are very clean and not too complex. If this is not the case or sample throughput is more important than mass accuracy, MALDI-TOF MS is the first choice. Another important advantage of ESI is its superior performance for the analysis of noncovalent complexes including DNA-protein interactions. 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

Fundamentally, introduction of a gaseous sample is the easiest option for ICP/MS because all of the sample can be passed efficiently along the inlet tube and into the center of the flame. Unfortunately, gases are mainly confined to low-molecular-mass compounds, and many of the samples that need to be examined cannot be vaporized easily. Nevertheless, there are some key analyses that are carried out in this fashion the major one i.s the generation of volatile hydrides. Other methods for volatiles are discussed below. An important method of analysis uses lasers to vaporize nonvolatile samples such as bone or ceramics. With a laser, ablated (vaporized) sample material is swept into the plasma flame before it can condense out again. Similarly, electrically heated filaments or ovens are also used to volatilize solids, the vapor of which is then swept by argon makeup gas into the plasma torch. However, for convenience, the methods of introducing solid samples are discussed fully in Part C (Chapter 17). 

Until 1981, mass spectrometry was limited, generally, to the analysis of volatile, relatively low-molecular-mass samples and was difficult to apply to nonvolatile peptides and proteins without first cutting them chemically into smaller volatile segments. During the past decade, the situation has changed radically with the advent of new ionization techniques and the development of tandem mass spectrometry. Now, the mass spectrometer has a well-deserved place in any laboratory interested in the analysis of peptides and proteins. 

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. 

Fast-atom bombardment (FAB) is an ionization technique that produces a protonated or deprotonated molecular ion, hence a molecular mass for the sample. It can be used for analysis of peptides up to m/z about 5000. 

ICPMS is uniquely able to borrow a quantitation technique from molecular mass spectrometry. Use of the isotope dilution technique involves the addition of a spike having a different isotope ratio to the sample, which has a known isotope ratio. This is usefiil for determining the concentration of an element in a sample that must undergo some preparation before analysis, or for measuring an element with high precision and accuracy. ... 

Fig. 7 gives an example of such a comparison between a number of different polymer simulations and an experiment. The data contain a variety of Monte Carlo simulations employing different models, molecular dynamics simulations, as well as experimental results for polyethylene. Within the error bars this universal analysis of the diffusion constant is independent of the chemical species, be they simple computer models or real chemical materials. Thus, on this level, the simplified models are the most suitable models for investigating polymer materials. (For polymers with side branches or more complicated monomers, the situation is not that clear cut.) It also shows that the so-called entanglement length or entanglement molecular mass Mg is the universal scaling variable which allows one to compare different polymeric melts in order to interpret their viscoelastic behavior. 

The broad pore size distribution of Sepharose makes it well apt for the analysis of broad molecular mass distributions of large molecules. One example is given by the method for determination of MWD of clinical dextran suggested in the Nordic Pharmacopea (Nilsson and Nilsson, 1974). Because Superose 6 has the same type of pore size distributions as Sepharose 6, many analytical applications performed earlier on Sepharose have been transformed to Superose in order to decrease analysis time. However, Sepharose is suitable as a first try out when no information about the composition of the sample, in terms of size, is available. 

Polyacrylic acid (pAA) homopolymers and related copolymers have become a commercially important class of water-soluble polymers. Acrylic acid polymers can range in molecular mass from less than 1000 Da to greater than 1,000,000 Da. A representative set of analysis conditions is... 

FIGURE 5.23 Electrospmy mass spectrum of the protein, aerolysin K. The attachment of many protons per protein molecule (from less than 30 to more than 50 here) leads to a series of m/z peaks for this single protein. The inset shows a computer analysis of the data from this series of peaks that generates a single peak at the correct molecular mass of the protein. (Adapted from Figure 2 in Mann, M., and Wilm, M., 1995. Trends in Biochemical Sciences 20 219-224.)... 

An intensely colored by-product of the photolysis reaction of methyl-2-azidobenzoate has been identified as the first known derivative of 3,3 -diazaheptafulvalene 70 (94LA1165). Its molecular mass was established by elemental analysis and mass spectroscopy as that of a formal nitrene dimer, whereas and NMR studies demonstrated the twofold symmetry as well as the existence of a cross-conjugated 14 7r-electron system in 70. Involving l-azido-2,3-dimethoxy-5,6-dimethoxycarbonylbenzene in thermal decomposition reactions, the azaheptafulvalene 71 could be isolated and characterized spectroscopically and by means of X-ray diffraction. Tliis unusual fulvalene can be regarded as a vinylogous derivative of azafulvalenes (96JHC1333) (Scheme 28). 

Figure 13,12 Illusti ation of the clean-up method, showing the analysis of an air sample (a) with and (b) without column switching. Details of the analytical conditions are given in the text. Reprinted from Journal of Chromatography, A 697, R R. Kootsti a and H. A. Herbold, Automated solid-phase exti action and coupled-column reversed-phase liquid cltromatogra-phy for the trace-level determination of low-molecular-mass carbonyl compounds in ak , pp. 203-211, copyright 1995, with permission from Elsevier Science.

The term titrimetric analysis refers to quantitative chemical analysis carried out by determining the volume of a solution of accurately known concentration which is required to react quantitatively with a measured volume of a solution of the substance to be determined. The solution of accurately known strength is called the standard solution, see Section 10.3. The weight of the substance to be determined is calculated from the volume of the standard solution used and the chemical equation and relative molecular masses of the reacting compounds. 

The complete analysis of alcohol sulfates is described in the Standard Methods of the International Organization of Standards (ISO) [200] and of the American Society for Testing and Materials (ASTM) [201]. These methods describe the analysis of inorganic sulfate content, chloride content, unsulfated matter, and water as well as other analytical values. Other ISO standards describe the analysis of sodium secondary alkyl sulfates [202], determination of pH [203], determination of water content [204,205], chlorides [206], total active matter in sul fated ethoxylated alcohols and alkylphenols [207], mean relative molecular mass in sulfated ethoxylated alcohols and alkylphenols [208], sulfate content... 

The TIC trace from this analysis, shown in Figure 5.5, exhibits a maximum at ca. 19 min, and a representative electrospray spectrum is illustrated in Figure 5.6. Transformation of the latter produces the spectrum presented in Figure 5.7 which indicates the presence of two species with relative molecular masses (RMMs) of 56 548.5 and 58 161.4 Da. These masses are lower than the value of 59.1 kDa calculated from previously obtained sequence information. 

Subsequent studies provided a wealth of information that appeared to support the hypothesis that the Fepr protein was a genuine [6Fe-6S]-containing protein. In a biochemical study (10) the elemental analysis was meticulously repeated, and, based on an assumed molecular mass of 52 kDa, the prismane protein was found to contain 6.3 Fe atoms, averaged over as many as nine different preparations. Again, no other metals than Fe were detected, suggesting that all... 

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