2D polyacrylamide gels

D) polyacrylamide gels. These types of experiments have been performed for more than twenty years to build databases of proteins expressed from certain cell or tissue types (Anderson and Anderson, 1996 O Farrell, 1975). Although this remains an important component of proteomics research, the field has expanded due to the development of additional technologies. Proteomics can be broadly divided into two areas of research (i) protein expression mapping, and (ii) protein interaction mapping. 

D-polyacrylamide gel electrophoresis) maps of protein mixtures is discussed. 2D PAGE is considered the classical and principal tool for protein separation—prior to mass spectrometry—to achieve the main goal of proteomics, that is, a comprehensive identification and quantification of every protein present in a complex biological sample that would allow analysis of an entire intact proteome (Wilkins et al., 1997 Righetti et al., 2001 Hamdan and Righetti, 2005). 

FIGURE 4.1 2D polyacrylamide gel electrophoresis (2D-PAGE) maps of protein mixtures. (See text for full caption.)... 

D polyacrylamide gel electrophoresis (2D PAGE) and MS are weU-established and the most commonly employed techniques in proteomics today. 2D PAGE, however, provides limited information of the total amount of proteins. Low-abimdance proteins and small peptides are not detected [1]. Additional methodologies and techniques in sample preparation, selective enrichment, high resolution separation, and detection need to be developed which would allow even higher resolution than 2D PAGE. Acceptable sensitivity to detect the low-abundance proteins is also still an issue. LC can address some of the above-mentioned... 

Several examples are known in which the separation itself was done by using a planar technique, and the detection by using direct visualisation. X-ray detection or mass spectrometry after transfer of the spots to another plane. In one example the separation was done by two-dimensional (2D) slab electrophoresis and then applied to 2D-TLC directly combined with mass spectrometry 111. -115]. The methods cannot be directly applied for 2D polyacrylamide gel electrophoresis because an essential portion (S-Ll f) of the polyacrylamide gel is water, and therefore a 9 x 1.1 cm plate of polyacrylamide contains 15 ml water 1116. This is the reason that the gel cannot be directly inserted into the high-vacuum source of the mass spectrometer. To avoid the complications of freeze-drying and other methods of removing the water and to keep the substances in the gel as well as the form of the gel, the separated substances should be transferred to a plate. Nitrocellulose was found to have the properties required and this method was used to detect bradykinin and dynorphine using SIMS (Secondary /on Mass Spectrometry). A detailed description is given for TLC-SIMS (115]. 

However, the exact number of proteins present in the archaeal ribosome is still unknown. The number quoted by various investigators, based on 2D polyacrylamide gel electrophoresis, depends on the resolving power of the gel system used. Recent studies by Casiano et al. [11] suggest that the Sulfolobus 508 ribosomal subunit could contain as many as 43 r-proteins, and work from our laboratory [12] on the structure of the Sulfolobus r-proteins and from Wittmann s laboratory [13] on Haloarcula (formerly Halobacterium) marismortui r-proteins indicates an increasing number of proteins present in the archaeal ribosome that are not present in the bacterial ribosome. It may well be that the archaeal ribosome contains more proteins than are present in the bacterial E. coll) ribosome, or the bacterial ribosome contains proteins not found in the archaeal ribosome (see section 6). 

Since all the r-proteins from both the large (L) and the small (S) ribosomal subunits of E. coli are sequenced and well characterized [106], homologous proteins from other organisms are named according to their E. coli counterpart. For example, proteins homologous to L23 of E. coli (Eco L23) will be called Sac L23 in Sulfolobus acidocaldarius, Hma L23 in Haloarcula marismortui and See L23 in Saccharomyces cerevisiae. In Table 2 (below), we have used this nomenclature to identify any r-protein that, from its sequence similarity, was equivalent to a known E. coli r-protein. Proteins which have no counterparts in E. coli (or where the sequence similarity is too weak to identify the relationship) are listed by the numbers described in the original publications and a trivial name. These numbers usually refer to the mobility of the protein in a 2D polyacrylamide gel. For example, HS15 is a protein from the small ribosomal subunit of H. marismortui which has no counterpart in E. co/j [107]. 

The primary amino acid sequence of polypeptide is routinely determined using the commercially available gas-liquid-phase sequencers [1, 2] and solid-phase sequencers [3, 4] based on the Mman degradation chemistry [.5j. These instruments can routinely obtain the primary amino acid sequence from 10 to 100 pmol of polypeptide. However, the need for higher sequencing sensitivity remains, as Kent et al. [6] have pointed out that rare proteins may only be present at the 30-300 fmol level on 2D-polyacrylamide gels. 

The capacity of the nanoparticles to adsorb proteins and to activate the complement in vivo after intravenous administration will influence the fate of the carrier and its body distribution. To approach this aspect, in vitro tests have been developed to investigate the profile of the type of serum proteins that adsorbed onto the nanoparticle surface after incubation in serum and to evaluate the capacity of the nanoparticles to induce complement activation. The analysis of the protein adsorbed onto the nanoparticle surface can be performed by 2D-polyacrylamide gel electrophoresis. This technique allows the identification of the proteins that adsorbed onto the nanoparticle surface. To evaluate modifications of the composition of the adsorbed protein with time, a faster method based on capillary electrophoresis can also be used. Finally, the activation of the complement produced by nanoparticles can be evaluated either by a global technique or by a specific method measuring the specific activation... 

Although few papers have yet been published in the new field of proteomics for parasitic diseases, efforts are tangible and should lead to important discoveries. The most widely used technology for carrying out proteomic analyses consists of two-dimensional (2D) polyacrylamide gels, which are studied and compared by powerful new image analysis softwares. 

IEF is the conventional method for protein separation in two-dimensional (2D) polyacrylamide gel electrophoresis and is considered one of the most powerful techniques available for separating proteins. However, IEF in gels is a time-consuming technique. Hjerten and Zhu, therefore, adapted IEF to fused-silica capillaries, so-called capillary IEF (cIEF), to minimize analysis times. No gels are used in cIEF instead, the capillary is filled with ampholytes in free solution to create the pH gradient. This technique is also called liquid-phase IEF. Additional advantages of cIEF are the potential for system automation and the possibility of on-line detection. 

Detection of radiolabeled proteins Mass spectrometry (electrospray ionization, matrix-assisted laser desorption, ionization) Gel electrophoresis (2D polyacrylamide gel electrophoresis)... 

Electrophoresis-based separation techniques have been widely used in pro-teomics studies. The most commonly used electrophoresis-based technique is gel electrophoresis (GE), which is a procedure for separating a mixture of molecules through a stationary material (gel) in an electrical field. The GE, especially two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE), is a powerful tool for protein separation. ... 

---