Protein separation profile

Figure 1. Separation profile on Sephacryl SlOO column of extracellular PG. 2.7 ml fractions were analyst for protein ( ) and reducing sugars released (A). Peaks I, II, III and IV correspond to PG activity expressed as pmol galacturonic acid released min V...

The ProteinChip System from Ciphergen Biosystems uses patented SELDI (Surface-Enhanced Laser Desorption/Ionization) ProteinChip technology to rapidly perform the separation, detection, and analysis of proteins at the femtomole level directly from biological samples. ProteinChip Systems use ProteinChip Arrays which contain chemically (cationic, anionic, hydrophobic, hydrophilic, etc.) or biochemically (antibody, receptor, DNA, etc.) treated surfaces for specific interaction with proteins of interest. Selected washes create on-chip, high-resolution protein maps. This protein mass profile, or reten-tate map of the proteins bound to each of the ProteinChip Array surfaces, is quantitatively detected in minutes by the ProteinChip Reader. 

FIGURE 15.3 Outline of experimental protocol used for ICAT differential protein expression profiling. Protein mixtures from two cell populations are labeled with light or heavy isotopic versions of a cleavable ICAT reagent. Labeled proteins are combined, subject to multidimensional separation by SCX, RP, and avidin affinity chromatography, then analyzed by tandem MS for peptide and protein identification. Based on the relative ratio of the two isotopically labeled peptides, a relative abundance of protein expression can be determined. 

A detailed examination of the affinity of SLPI for the heparinized capillary was next made using a stepwise elution (from 0.1 to 0.9 M NaCl) (Fig. 11). SLPI eluted from the capillary with 0.2 M NaCl. This agreed well with results obtained by traditional affinity chromatography on a heparin-Sepharose matrix. The ACE method has the unique advantages over traditional affinity chromatography in that it requires much smaller quantities of protein and afforded better separation profiles. 

To analyze the differenees of protein expression profiles between caneer tissues and corresponding non-caneerous tissues, the proteomic differential display method was used. In this method, 2-DE and MS were used to identify the proteins. We first separated proteins from caneer tissues and eorresponding non-cancerous tissues by 2-DE. Then the protein spots of the samples from eaneer tissues were compared to the spots of the samples from corresponding non-eaneerous tissues by using software for proteomie differential display. 

Figure 6.2 Identification of proteins separated by 2-DGE. (A) Base-peak profile from the capillary HPLC separation from a selected protein spot. (.B) Mass spectrum of the second major chromatographic peak (residue 60-70). (C) LC/MS/MS product ion spectrum of m/z 451.3. (Reprinted with permission from Amott et al., 1995. Copyright 1995 American Chemical Society.)...

Quantitation Once protein expression profiling activities characterize qualitative features, the attention turns to delineating protein interactions and mechanistic pathways responsible for disease. These studies also require rapid sequence determination/confirmation combined with accurate and sensitive quantitative analysis. The quantitation approaches would allow for direct comparison of protein amounts (absolute or relative) from a variety of cellular states. Because of the reasons stated previously, quantitative applications are likely to be less dependent on 2-DGE and rely primarily on formats that involve specific purification and/or chromatographic separation with mass spectrometry. 

Proteins and Amino Acids Total protein in food and feed samples is commonly determined by Kjeldahl (acid digestion/titration) or Dumas (pyrolysis) or elemental analysis.14 FIPLC can separate major proteins and furnish protein profiles and speciation information. HPLC can be used to further characterize specific proteins via peptide mapping and amino acid sequence analysis. HPLC modes used for protein include IEC, SEC, RPC, and affinity chromatography with typical UV detection at 215 nm or MS analysis. Details on protein separations are discussed in the life sciences section. 

Figure 4. Calculated elution profile to simulate a protein separation on Sephadex G-7S using equation (12). This simulation is based on parameter values for a 30 x 1.5 cm column with a flowrate of 1.77 ml/min and a longitudinal diffusivity of 0.01 cm2/min. The ratio of mobile phase volume to pore volume was 0.9, and the sample volume was 0.17 ml. Capacity factors for each of the solutes are 0, 0.5, and 1.1, respectively.

The ideal solubilisation procedure for 2-DE would result in the disruption of all non-covalently bound protein complexes and aggregates into a solution of individual polypeptides (Dunn and Gorg, 2001). If this is not achieved, persistent protein complexes in the sample are likely to result in new spots in the 2-D profile, with a concomitant reduction in the intensity of those spots representing the single polypeptides. In addition, the method of solubilisation must allow the removal of substances, such as salts, lipids, polysachharides and nucleic acids that can interfere with the 2-DE separation. Finally, the sample proteins must remain soluble during the 2-DE process. For the foregoing reasons sample solubility is one of the most critical factors for successful protein separation by 2-DE. 

It is therefore clear that until these alternative approaches mature into robust techniques for quantitative protein expression profiling, 2-DE will remain the separation work-horse in many proteomic investigations. This technique has the capacity to support the simultaneous analysis of the changes in expression of hundreds to thousands of proteins and as such it remains unrivalled as an open protein expression profiling approach. 

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