Mass spectrometric characterization of proteins transferred from polyacrylamide gels to membrane filters

Abstract

In the 1990s, a technique was developed to transfer proteins from electrophoresis gels onto poly(vinylidene difluoride) (PVDF) membranes, digest the proteins on the membranes with proteases such as trypsin and analyze the resulting peptides on the membranes directly by mass spectrometry to identify the proteins. This technique, based on gel electrophoresis, is particularly useful for analyzing protein isoforms, splicing variants and post-translationally modified proteins. Previously, the low ionization efficiency of peptides immobilized on the membranes often rendered this technique useless. However, this technique has been improved by the use of PVDF membranes with a small pore size, which has enabled highly efficient and effective electroblotting and mass spectrometric analyses. Here, the advantage of this technique is discussed. Proteomic analyses begin with protein expression profilingProteome research involves the comprehensive analysis of protein expression profiles, the function and functional networks of proteins, and the relationship between proteins and diseases. Upgraded protein databases, and highly sensitive, accurate and high-throughput analytical techniques, such as mass spectrometry (MS), have greatly facilitated and accelerated the development of proteome research.Two standard methods, the protein shotgun method [1] and the 2D electrophoresis (2DE)-MS ⁄ MS method [2], are commonly used in the first step of proteome analysis. In the shotgun method, a sample containing a number of proteins is digested with proteases such as trypsin, which has comparatively high substrate specificity. These digested proteins are separated by liquid chromatography (LC), analyzed by MS ⁄ MS and then identified based on their sequence information. In contrast, in the 2DE-MS ⁄ MS method, most proteins are separated by 2DE, protease-digested in gels and then analyzed by MS [3]. Next, the proteins are identified based on peptide mass fingerprints or on sequence information obtained by MS ⁄ MS and database searches [4].Both methods can profile the expression of a number of proteins. However, the shotgun method has been more frequently used for recent proteomic analyses. The shotgun method typically identifies more proteins than 2DE-MS ⁄ MS. In addition, the shotgun method can be easily automated and therefore does not require professional skills for the analysis. In contrast, 2DE is laborious, and it is not easy for beginners to obtain reproducible results, particularly with the 'in-gel' digestion of proteins after electrophoresis. However, the shotgun method cannot completely replace 2DE-MS ⁄ MS for proteomic studies because 2DE-MS ⁄ MS has several advantages over the shotgun method, as described below.

A Study of Rotational Ultrafiltration System for Fructose Recovery from Glucose Fermentation Process

“…Lee and Rose [8] and Lewandowicz et al [9] used 0.2 µm film membrane distillation to separate and remove yeast, indicating that the size of yeast should be greater than 0.2 µm. Other studies by Sung et al [10] and Ino and Hirano [11] used 0.1 µm and 0.45 µm pore size ceramic and PVDF membrane to separate fermented residue. Youn et al [12] used 0.01 µm pore size membranes with molecular weight cut-off greater than 30,000 to filter reconstituted apple juice, yielding a filtrate containing 99% sugar, showing that fructose molecules are smaller than 10 nm.…”

On-Membrane Digestion Technology for Muscle Proteomics

“…Protein transfer technology has also been integrated into modern mass spectrometry-based proteomics [21], especially exploiting the efficient onmembrane proteolytic digestion of electro-blotted molecules for swift protein identification [22]. Proteomics is a technology-driven and hypothesisgenerating approach that combines established biochemical and protein chemical methods for the comprehensive survey of large protein populations [23][24][25].…”

“…In-gel trypsination is the most frequently used method to generate peptides from gel electrophoretically separated protein mixtures [34,35]. However, this technique does sometimes not result in an efficient proteolysis of distinct protein species for their subsequent identification by mass spectrometry, which necessitates the application of alternative methods such as on-membrane trypsination [21,22]. Since membrane-bound proteins appear to be more prominently exposed to proteases, the controlled digestion of proteins transferred from gel bands or spots to membrane sheets is advantageous when studying certain protein species.…”