Principles of Protein Labeling Techniques.

Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity, and simplification of detection workflows. Proteins can be labeled during cell growth by incorporation of amino acids containing different isotopes, or in biological fluids, cells or tissue samples by attaching specific groups to the ε-amino group of lysine, the N-terminus, or the cysteine residues. The principles and the modifications of the different labeling approaches on the protein level are described; benefits and shortcomings of the methods are discussed.

Detection and Identification of Low-Abundant Proteins Using HPE Gels, Fluorescent Stains, and MALDI-ToF-ToF-MS.

Two-dimensional electrophoresis as a complementary approach to gel-free proteomic methods possesses the ability to separate physiologically important isoforms of proteins in an unbiased manner. Frequently, those isoforms are low-abundant regulators, and therefore, detection and identification of low-abundant proteins is highly necessary to exploit this advantage. We describe an experimental sequence of classical operations to process gels but optimized them, in order to identify each detectable protein spot on gel.

2D gel-based Proteomics: there's life in the old dog yet.

In the nowadays mainly mass spectrometry-driven world of Proteomics, one can frequently read and hear that 2D gel-based electrophoresis methods are no longer used for proteome analysis. This short review is a compilation of facts why the gel-based workflow is not dead.

Principles of protein labeling techniques.

Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity, and simplification of detection workflows. Proteins can be labeled during cell growth by incorporation of amino acids containing different isotopes, or in biological fluids, cells or tissue samples by attaching specific groups to the ε-amino group of lysine, the N-terminus, or the cysteine residues. The principles and the modifications of the different labeling approaches on the protein level are described; benefits and shortcomings of the methods are discussed.

Looking at proteins from two dimensions: a review on five decades of 2D electrophoresis.

Separating proteins according to two different gel-electrophoretic methods not only increases the resolution power for highly complex samples when compared to one-dimensional separations, but is also a valuable tool for protein and protein complex characterization. There are a number of different electrophoresis methods which can be combined. The combination of isoelectric focusing under denaturing conditions and SDS polyacrylamide gel electrophoresis delivers the highest resolution of all bio-analytic techniques. This is a short review on the history and state of the art of two-dimensional electrophoresis methods, and contains some practical tips for high resolution 2D electrophoresis, which are based on several decades of experience with this method.

Simplification and improvement of protein detection in two-dimensional electrophoresis gels with SERVA HPE™ lightning red.

A new fluorescent amino-reactive dye has been tested for both labelling proteins prior to electrophoretic separations and between the two steps of two-dimensional electrophoresis. A series of experiments showed, that the labelling of lysines with this dye is compatible with all standard additives used for sample preparation, including reducing substances and carrier ampholytes. Using this dye for pre-labelling considerably simplifies the electrophoresis and detection workflow and provides highly sensitive and quantitative visualisation of proteins.

The new horizon in 2D electrophoresis: new technology to increase resolution and sensitivity.

A principally new type of an electrophoresis setup for the second dimension of 2DE named HPE (high performance electrophoresis) has recently become available that provides excellent reproducibility much superior to traditional 2DE. It takes up ideas from early beginnings of 2DE which could not be satisfactory realized at that time. The new HPE system is in contrast to all other established systems a horizontal electrophoresis that employs a new type of precast polyacrylamide gels on film-backing and runs on a multilevel flatbed electrophoresis apparatus. In a systematic approach we compared its features to traditional 2DE for the cytosolic proteome of Bacillus subtilis. Not only the reproducibility is enhanced, but also nearly all qualitative parameters as resolution, sensitivity, the number of protein spots (25% more), and the number of different proteins (also additional 25%) are markedly increased. More than 200 proteins were exclusively found in HPE. This new electrophoresis system does not use buffer tanks. No glass plates are needed. Therefore handling of gels is greatly facilitated and very simple to use even for personnel with low technical skills. The new HPE system is technically at the beginnings and further development with increased performance can be expected.

Isoelectric focusing and two-dimensional gel electrophoresis.

By far the highest resolution of all separation techniques for intact proteins in a single analytical run continues to be by the combination of isoelectric focusing (IEF) and SDS-PAGE, originally introduced by O'Farrell [(1975). J. Biol. Chem.250, 4007-4021]. This analytical platform has seen a number of significant advances and applications over the past 25years, including reproducibility using immobilized pH gradient (IPG) strips [Bjellqvist et al. (1982). J. Biochem. Biophys. Methods6, 317-339.], resolution in alkaline IEF using hydroxyethyldisulfide (HED) [Olsson et al. (2002). Proteomics2, 1630-1632], and quantification for differential expression proteomics on intact proteins on a global scale [DIGE; Unlu et al. (1997). Electrophoresis18, 2071-2077]. These major improvements will be highlighted in this chapter alongside the principle and theory of 2D gel electrophoresis, as well as detailed methods for general 2D gel electrophoresis best use protocols.

The current state of the art in high-resolution two-dimensional electrophoresis.

The study of the "proteomes" of human cells, tissues, and body fluids is a big challenge, and several highly sophisticated workflow approaches are pursued to achieve as comprehensive information as possible. Initially proteome analysis was exclusively based on the gel-based workflow, employing two-dimensional electrophoresis of protein extracts followed by mass spectrometry of the tryptic peptide digests of protein spots. Meanwhile several additional proteomics workflows are applied, which are mostly based on separation and analysis of tryptic peptides without separating the protein mixture. However, direct information on quantitative and qualitative changes of protein expressions can only be obtained by methods operating on the protein level, no other method can replace two-dimensional electrophoresis. In this review we compile the different techniques of high-resolution two-dimensional electrophoresis and their further developments to increase the degree of reliance of the method.

Difference gel electrophoresis based on lys/cys tagging.

Before separation, proteins of different biological samples are labeled with different fluorescent dyes, the CyDye DIGE Fluors. Currently three dyes with spectrally different excitation and emission wavelengths are available. This allows labeling up to three different samples, and coseparating them in one gel. The dyes can either be attached to the epsilon-amino side group of the lysine without derivatization of the polypeptides or to the cysteines after reduction of the disulfide bonds. For lysine labeling a so called minimal labeling approach is performed: only a low-ratio dye: protein is applied in order to prevent multiple labels per protein. Although only 3% of the proteins are tagged, the sensitivity of detection is comparable with the sensitivity of a good quality silver staining. The dyes are matched for size and charge to obtain migration of differently labeled identical proteins to the same spot positions. The spot pattern achieved with minimal labeling is similar to the pattern obtained with poststained gels. When cysteine tagging is applied, all cysteine moieties are labeled. This modification of the method affords extraordinarily high sensitivity of detection. However, because of multiple labeling, the resulting pattern will look different from nonlabeled or minimal labeled samples. The labeled samples are mixed together before they are applied on the gel of the first dimension. After separation the gels are scanned with the multifluorescent imager at the different wavelengths. Up to three images of comigrated protein mixtures are compared and evaluated from each gel. This multiplexing technique allows the application of an internal standard for each protein in a complex mixture: One of the labels is applied on a mixture of the pooled aliquots of all samples of an experiment. By coseparating this mixture with each gel an internal standard is created for reliable and reproducible detection and assessment of changes of protein expression levels. Image analysis is performed with special software, which allows codetection of protein spots across the different samples and the internal standard.

Frequently made mistakes in electrophoresis.

In spite of text books, instrument manuals, product instructions, and web tutorials there are a number of erroneous protocols around, which lead repeatedly to issues during electrophoresis runs and to inadequate results. The relatively low resolution and short running time of miniformat systems often conceals these issues. However, in high-resolution 2-D electrophoresis in large format gels, one of the most important separation methods in Proteomics, the consequences of these mistakes become more obvious.

Preparation of plant samples for 2-d electrophoresis.

Plant protein samples are very difficult to extract and prepare for 2-D electrophoresis, because polyphenols can build irreversible complexes with the proteins, the protein concentrations are relatively low, polysaccharides and lipids can cause severe disturbances in the 2-D gel.

Sensitive, quantitative, and fast modifications for Coomassie Blue staining of polyacrylamide gels.

In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue staining is still the most widely used protein detection technique for proteins separated by polyacrylamide gel electrophoresis. There are several reasons: Low price, Visible with the eye, Desk top scanners can be employed for image acquisition, Better for quantitative analysis than silver staining, Possible modifications for fast or highly sensitive staining, Mass spectrometry compatible.

Protein detection methods in proteomics research.

In proteomics research chemical as well as physical methods are used to detect proteins subsequently to their separation. Physical methods are mostly applied after chromatography. They are either based on spectroscopy like light absorption at certain wavelengths or mass determination of peptides and their fragments with mass spectrometry. Chemical methods are used after two-dimensional electrophoresis and employ staining with organic dyes, metal chelates, fluorescent dyes, complexing with silver, or pre-labeling with fluorophores. In some cases autoradiography is still used. Since all of these techniques are very different in terms of sensitivity, their usefulness for quantitative determinations varies significantly. This review will describe the various protein detection methods applied to electrophoresis gels.