Sample preparation membrane proteins

In one study by Hood et al., 282 of 1153 identified proteins were identified by at least 2 unique tryptic peptides from FFPE prostate cancer (PCa) tissue.9 According to the gene ontology classification of the proteins identified, -65% of proteins were predicted to be intracellular proteins, while -50% of the total human proteome is predicted to be located in the intracellular compartment. Additionally, 20% of the proteins identified in the PCa tissue were classified as membrane proteins, which is significantly less than the predicted 40% for the human proteome. This relative disparity is not unexpected, considering the Liquid Tissue sample preparation kit lacks specific protocols for membrane protein extraction. The Liquid Tissue method has also been used for proteomics studies of a variety of FFPE tissue samples, including pancreatic tumors,28 squamous cell carcinoma,4 and oral human papillomavirus lesions.27... 

The last sample preparation method for IMS is the transfer of a tissue section onto the PVDF membrane. Proteins in the section can be transferred onto the PVDF membrane and then analyzed on the membrane. The advantage of this method is that the enzyme can be digested for MS" measurement, because the information on protein localization in the organization is fixed on the membrane.5,20 This technique can denature, reduce, and digest the proteins in the tissue section efficiently and remove the salt from the tissue. This increases the efficiency with which biological molecules are ionized, making it possible to obtain sensitive mass imaging spectra. 

Retention of a protein or protein activity after 105,000y, 1 hr Chromatography on gel filtration columns with large pore sizes Electron microscopy—however, sample preparation may partially reconstitute membranes Decrease in solution turbidity, which may be detected by a diminution in light scattering or an enhancement in light transmission Diffusion of membrane lipids as assayed by nuclear magnetic resonance and electron spin resonance... 

The limited solubility of membrane proteins and related polypeptides in aqueous mobile phases can also cause problems. These could be solved, e.g., by adding guanidine hydrochloride (6 M) or urea (8 M) to the portion of initial eluent used for sample preparation 69). The urea was always eluted in the breakthrough volume of the column. Thus, the retained hydrophobic polypeptides might have been temporarily precipitated upon the column. Collagen chains, dissolved in 0.5 M acetic acid, were successfully separated by RP-HPLC through gradients of 0.1 M TFA/acetonitrile 70> or (0.05 M ammonium bicarbonate + TFA)/ tetrahydrofuran 57>. 

The protocol involves a classical SDS-PAGE (10% polyacrylamide) run, followed by transfer onto a Western blot membrane and immunodetection with an anti-pIII antibody. Nevertheless, special care must be taken during sample preparation, because phages are very stable and difficult to denature. The protocol is similar to typical SDS-PAGE sample preparation, except that / -mer cap toe thanol should be replaced by fresh dithiothreitol (DTT, 5 mM final concentration), and the samples should be boiled in a water bath for at least 15 min. Moreover, because the pIII-fusion protein is a minor component of the virion, a large amount of phages should be loaded onto the gel, typically around 1012 phages per lane. 

The technique of ultrafiltration for serum sample preparation has been studied by several investigators (H7, HIO, K9, S19, V4, V5). In this technique the serum sample is passed through an exclusion membrane which exhibits a 95% retention for compounds with MW > 25,000. Thus, low-molecular-weight constituents pass through the membrane, whereas proteins or protein-bound constituents are retained. Hartwick ct al. (HIO) reported excellent recoveries for xanthosine, inosine, guanosine, theobromine, theophylline, and caffeine. Tryptophan showed poor quantitative recovery in accordance with the findings of previous investigators (K33, 01). 

It should be obvious from the above that membrane proteomics strictly follows Murphy s law. This is due to the fact that there is a mutual exclusion, for physicochemical reasons, between on the one hand the conditions that must be used to solubilize in water all membrane proteins, including the most hydrophobic ones, and thus give a fair representation of the protein population in the sample, and on the other hand the conditions prevailing in the high resolution peptide separation methods. On top of this problem, there is a second problem linked to the membrane versus aqueous phase volume in many subcellular preparations, which makes transmembrane proteins rare compared to water-soluble proteins. 

---