Molecular mass of proteins

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

Figure 50-1. Relative dimensions and approximate molecular masses of protein molecules in the blood (Oncley).

In the example shown in Figure 1.23 using the peaks at m/z 939.2 and 1372.5 (j = 6), we obtain z = 6(1372.5 - 1.0073)/(1372.5 - 939.2) = 19 and we can number all the peaks measured according to the number of charges. M can be calculated from their mass. The average value obtained from all of the measured peaks is 17 827.9 Da with a mean error of 2.0 Da. This technique allowed the determination of the molecular masses of proteins above 130 kDa with a detection limit of about 1 pmol using a quadrupole analyser. 

Blue copper oxidase Amino acids Molecular mass of protein (kDa) Molecular mass of whole enzyme (kDa) Carbohydrate content ) IP Reference... 

The molecular mass of protein and peptide samples was determined by electrospray ionization mass spectrometry using a Perkin-Elmer Sciex API 100 mass spectrometer. The sample was introduced either by infusion or by on-line liquid chromatography/mass spectrometry (LC/MS) using a splitter. The data were obtained by scanning from 450 to 2000 Da with a scan time of 5 s and a step size of 0.25 Da with 1.0 ms dwell time per mass step. The molecular mass of the sample was obtained using the software provided by the instrument manufacturer. 

We have also determined the upper limit of deuterium incorporation in HCA II. For this purpose milligram quantities of H labeled protein were produced in defined media containing 98.8% D2O and [ Hs, 98%] sodium acetate as the sole carbon source using the optimized procedures outlined above. To quantitate the level of deuterium incorporation, we analyzed the molecular mass of purified HCA II by mass spectrometry. The molecular mass of fully protonated HCA II was measured to be 29102 +/- 2.4 (theoretical mass = 29098.9). At low pH the protein contains 2018 protons therefore, one would predict a theoretical mass increase of 2030.5 mass units upon complete deuteration. The molecular mass of protein produced in 98.8% D2O and ["H3, 98%] sodium acetate was measured to be 31133 +1-13, an increase of 2034 +/- 15 mass units, indicating above 96% deuterium incorporation. 

M. Ibrahim, Z. Gongwei, Z. Junjie, Determination of diffusion coefficients of proteins by flow injection analysis and its application to estimation of molecular masses of proteins, Instrum. Sci. Technol. 26 (1998) 333. 

Williams, K. and Mozdzer, T. Determination of molecular masses of proteins in solution implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory, http //info.med.yale.edu/wmkeck/biophysics/ Presentation 04 01 02 final.pdf (2003). 

In the field of research methods probably the most important application of protein surfactant complexation is in the technique of polyacrylamide gel electrophoresis in the presence of SDS, the so-called SDS-PAGE technique, used for the analysis and estimation of molecular masses of protein subunits [22], Protein subunit-SDS complexes are formed from proteins reduced by /3-mercaptoethanol to remove disulphide bonds. The binding of SDS to the polypeptide chains oc-... 

The molecular masses of proteins range up to 2,750 kDa for the beta-lipoproteins, and the dynamic ranges for the plasma proteins range from millimoles per liter for albumin to femtomoles for the proteins in lower abundance. The molecular size of the individual protein is an important factor in determining its distribution and transport by the active and passive mechanisms of the body. Some proteins move freely in the extracellular and intravascular spaces, while other intracellular proteins are released only after cell damage. Some of the major plasma proteins are listed in Table 8.1, together with the broad protein fractions designated by their simple electrophoretic mobilities. 

Use MS data to determine relative molecular masses of proteins and other (macro)molecules... 

SDS-CGE Molecular size Estimation of relative molecular mass of protein (10-200kDa) and purity and stability of peptides and proteins SDS-PAGE... 

The identity of peptides and proteins can be determined by specihc activity assays, determination of amino acid composition and sequence, and assessment of such physico-chemical parameters as molecular mass and p7. Several CE techniques have been used for the identity of peptides and proteins, which include peptide mapping by CZE or CEC, CIEF for the determination of protein s p7, SDS-CGE for the determination of relative molecular masses of proteins, and CE-MS for direct molecular mass assignment of peaks separated by CE. 

MALDI-TOF and MALDI-TOF/TOF are straightforward techniques, and a large number of samples can be analyzed rapidly, particularly for masses in the 500 Da to 5 kDa mass range, e.g., to obtain mass maps (MS) and sequences (MS/ MS) of tryptic peptides that have been spotted on plates robotically. However, the lack of resolution and accuracy are limiting when analyzing proteins in the linear mode. Accordingly, MALDI-TOF cannot be used to assess small changes in the molecular mass of proteins, such as those that are due to phosphorylation or when a small molecule is added to a protein. 

Fig. 5. NaDodSO -PAGE of in vitro translation products of hTRP (lane 1) and rTRa vl (lane 2). The pGEM3 expression vector, T7 RNA polymerase and reticulocyte lysate was used as suggested by the supplier (Promega). Molecular masses of protein standards are indicated on the left.

Traditionally, the molecular mass of proteins has been estimated with SDS-PAGE, in which the migration pattern of a protein is compared to a set of known mass proteins. The accuracy of this procedure is, however, very poor (5 to 20%) and mitigates against its use in the applications pointed out above. More accurate mass values are required to distinguish a mutation between Asp and Asn, Asn and lle/Leu, Glu and Gin, and Lys and Glu/Gln. Currently, ESI and MALDl, combined with Q-TOF, orbitrap, and FT-ICR, have become a standard protocol to determine the molecular mass of intact proteins with an accuracy within 0.01%. 

Aqueous SEC was first reported in 1959 by Porath and Flodin [1]. They separated proteins and salts according to molecular size by using cross-linked dextran gels. Since then it has been widely employed, especially in the field of biochemistry, for various purposes such as purification of proteins and nucleic acids, estimation of molecular masses of proteins and determination of molecular mass distributions of polysaccharides. In addition, it has been a powerful tool for the determination of molecular mass distributions of water-soluble synthetic polymers since high-performance aqueous SEC was realized in 1978 by the development of semirigid microparticulate macroporous supports based on hydrophilic synthetic polymers [2-4]. 

Buffers containing 0.1% sodium dodecyl sulphate (SDS) [3j2], 6 M guanidine hydrochloride [33] or 7M urea [34] are also used for the separation of proteins. These solvents cause denaturation of proteins. When proteins are separated in a denatured state, they are eluted almost exactly according to their molecular mass. It is therefore possible to estimate molecular masses of proteins from elution volumes by utilizing a molecular mass calibration curve established with some standards of known molecular mass. Figure 7.16 is an... 

Another possibility is to use a capillary gel electrophoretic method that is nowadays a routinely and commercially available method for the determination of the molecular mass of proteins/polypeptides. This method can also be used for the separation of collagen chains and their polymers. For example, this procedure is described in the literamre for the separation of collagen type I a-chains and chain polymers p (dimers), and y (trimers), and also chain polymers of related molecular mass 300,(X)0 and higher (typically in the study of the formation of crosslinks). Besides commercially available kits, another option is to use fused-silica or poly vinylalcohol-coated capillaries filled with non-cross-linked polyacrylamide or hydroxyl-propylmethylcellulose in a 50 vaM Tris-glycine buffer (pH 8.8) or phosphate buffer (50 vaM, pH 2.5) (Table 1). 

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis has become one of the most used techniques for resolving and determining the apparent molecular mass of protein subunits. The SDS solubilizes insoluble proteins, making possible the analysis of otherwise insoluble mixtures. Here we describe the discontinuous gel system of Laemmli (1970) as well as a high-gel-density urea procedure for the separation of low-molecular-weight proteins (Schagger and von Jagow, 1987). 

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