Proteins determining precise mass

Figure 2.4. Peptide fingerprinting by MALDI-TOF mass Spectrometry. Proteins are extracted and separated on by 2D gel electrophoresis. A spot of interest is excised from the gel, digested with trypsin, and ionized by MALDI. The precise mass of proteolytic fragments is determined by time-of- flight mass spectrometry. The identity of the protein is determined by comparing the peptide masses with a list of peptide masses generated by a simulated digestion of all of the open reading frames of the organism.

Mass spectrometry can be used to determine the precise mass of a protein. Because of their large size and low volatility, proteins require specialized mass spectral techniques, such as electrospray ionization spraying a charged, heated stream of droplets into the evacuated source chamber. The solvent evaporates to leave ions of the compound to be analyzed. 

The strategy for bottom-up protein sequencing using mass spectrometry starts with the precise determination of the molecular mass of that protein using MALDI or ESI. This result allows one to verify the sequence that is determined ultimately and also to judge the homogeneity of the sample. The protein then is subjected to reduction and alkylation of the cysteine bridges. Determination of the molecular mass allows one to determine the number of cysteines present in the protein. 

The Mass of a Protein Can Be Precisely Determined by Mass Spectrometry... 

Techniques such as matrix-assisted laser desorption and ionization (MALDI) and electrospray ionization (ESI) allow the generation of ions of proteins and peptides in the gas phase. The mass of such protein ions can be determined with great accuracy and precision. Masses determined by these techniques act as protein name tags because the mass of... 

Enzymes are excellent catalysts for two reasons great specificity and high turnover rates. With but few exceptions, all reac tions in biological systems are catalyzed by enzymes, and each enzyme usually catalyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular struc ture of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (lU) of an enzyme is defined as the amount capable of producing one micromole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. 

The molecular mass of the protein was redetermined by infusing a 5-10 pmolp.l solution of the protein in 50% aqueous acetonitrile containing 0.2% formic acid at a flow rate of 6 p,lmin into an electrospray source. The scan rate employed on the mass spectrometer was from m/z 60 to m/z 1800 in 12 s. This is a relatively slow scan speed which will lead to a more precise molecular weight determination. Scan speeds of this order may be, and indeed should be, utilized for infusion experiments if sufficient sample is available but it is unlikely to be feasible when chromatographic separations, particularly those involving capillary columns, are employed because of the restriction imposed by the chromatographic peak width (see Section 3.5.2.1 above). 

Demmelmair, H. and Schmidt, H. L. (1993) Precise 813C determination in the range of natural abundance on amino acids from protein hydrolysates by gas chromatography isotope ratio mass spectrometry. Isotopenpraxis 29, 237 250. 

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). 

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