7,453 research outputs found
Power and limitations of electrophoretic separations in proteomics strategies
Proteomics can be defined as the large-scale analysis of proteins. Due to the
complexity of biological systems, it is required to concatenate various
separation techniques prior to mass spectrometry. These techniques, dealing
with proteins or peptides, can rely on chromatography or electrophoresis. In
this review, the electrophoretic techniques are under scrutiny. Their
principles are recalled, and their applications for peptide and protein
separations are presented and critically discussed. In addition, the features
that are specific to gel electrophoresis and that interplay with mass
spectrometry (i.e., protein detection after electrophoresis, and the process
leading from a gel piece to a solution of peptides) are also discussed
Two-dimensional gel electrophoresis in proteomics: A tutorial
Two-dimensional electrophoresis of proteins has preceded, and accompanied,
the birth of proteomics. Although it is no longer the only experimental scheme
used in modern proteomics, it still has distinct features and advantages. The
purpose of this tutorial paper is to guide the reader through the history of
the field, then through the main steps of the process, from sample preparation
to in-gel detection of proteins, commenting the constraints and caveats of the
technique. Then the limitations and positive features of two-dimensional
electrophoresis are discussed (e.g. its unique ability to separate complete
proteins and its easy interfacing with immunoblotting techniques), so that the
optimal type of applications of this technique in current and future proteomics
can be perceived. This is illustrated by a detailed example taken from the
literature and commented in detail. This Tutorial is part of the International
Proteomics Tutorial Programme (IPTP 2)
Improved mass spectrometry compatibility is afforded by ammoniacal silver staining
Sequence coverage in MS analysis of protein digestion-derived peptides is a
key issue for detailed characterization of proteins or identification at low
quantities. In gel-based proteomics studies, the sequence coverage greatly
depends on the protein detection method. It is shown here that ammoniacal
silver detection methods offer improved sequence coverage over standard silver
nitrate methods, while keeping the high sensitivity of silver staining. With
the development of 2D-PAGE-based proteomics, another burden is placed on the
detection methods used for protein detection on 2-D-gels. Besides the classical
requirements of linearity, sensitivity, and homogeneity from one protein to
another, detection methods must now take into account another aspect, namely
their compatibility with MS. This compatibility is evidenced by two different
and complementary aspects, which are (i) the absence of adducts and artefactual
modifications on the peptides obtained after protease digestion of a protein
detected and digested in - gel, and (ii) the quantitative yield of peptides
recovered after digestion and analyzed by the mass spectrometer. While this
quantitative yield is not very important per se, it is however a crucial
parameter as it strongly influences the S/N of the mass spectrum and thus the
number of peptides that can be detected from a given protein input, especially
at low protein amounts. This influences in turn the sequence coverage and thus
the detail of the analysis provided by the mass spectrometer.Comment: website publisher http://www.interscience.wiley.co
The Whereabouts of 2D Gels in Quantitative Proteomics
Two-dimensional gel electrophoresis has been instrumental in the development
of proteomics. Although it is no longer the exclusive scheme used for
proteomics, its unique features make it a still highly valuable tool,
especially when multiple quantitative comparisons of samples must be made, and
even for large samples series. However, quantitative proteomics using 2D gels
is critically dependent on the performances of the protein detection methods
used after the electrophoretic separations. This chapter therefore examines
critically the various detection methods (radioactivity, dyes, fluorescence,
and silver) as well as the data analysis issues that must be taken into account
when quantitative comparative analysis of 2D gels is performed
Simple, Time-Saving Dye Staining of Proteins for Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis Using Coomassie Blue
A fixation-free and fast protein-staining method for sodium dodecyl sulfate–polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye stain does not contain any amount of acid and methanol, such as phosphoric acid. Considering the speed, simplicity, and low cost, the dye stain may be of more practical value than other dye-based protein stains in routine proteomic research
Evaluation of four different strategies to characterize plasma membrane proteins from banana roots
Plasma membrane proteins constitute a very important class of proteins. They are involved in the transmission of external signals to the interior of the cell and selective transport of water, nutrients and ions across the plasma membrane. However, the study of plasma membrane proteins is challenging because of their poor solubility in aqueous media and low relative abundance. In this work, we evaluated four different strategies for the characterization of plasma membrane proteins from banana roots: (i) the aqueous-polymer two-phase system technique (ATPS) coupled to gelelectrophoresis (gel-based), and (ii) ATPS coupled to LC-MS/MS (gel free), (iii) a microsomal fraction and (iv) a full proteome, both coupled to LC-MS/ MS. Our results show that the gel-based strategy is useful for protein visualization but has major limitations in terms of time reproducibility and efficiency. From the gel-free strategies, the microsomal-based strategy allowed the highest number of plasma membrane proteins to be identified, followed by the full proteome strategy and by the ATPS based strategy. The high yield of plasma membrane proteins provided by the microsomal fraction can be explained by the enrichment of membrane proteins in this fraction and the high throughput of the gel-free approach combined with the usage of a fast high-resolution mass spectrometer for the identification of proteins
Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis
Mitochondria are complex organelles, and their proteomics analysis requires a
combination of techniques. The emphasis in this chapter is made first on
mitochondria preparation from cultured mammalian cells, then on the separation
of the mitochondrial proteins with two-dimensional electrophoresis (2DE),
showing some adjustment over the classical techniques to improve resolution of
the mitochondrial proteins. This covers both the protein solubilization, the
electrophoretic part per se, and the protein detection on the gels, which makes
the interface with the protein identification part relying on mass
spectrometry
Silver Staining of 2D Electrophoresis Gels
Silver staining is used to detect proteins after electrophoretic separation
on polyacrylamide gels. It -combines excellent sensitivity (in the low nanogram
range) with the use of very simple and cheap equipment and chemicals. For its
use in proteomics, two important additional features must be considered,
compatibility with mass spectrometry and quantitative response. Both features
are discussed in this chapter, and optimized silver staining protocols are
proposed.Comment: arXiv admin note: substantial text overlap with arXiv:0904.353
Effective detection of proteins following electrophoresis using extracts of locally available food species
Procedures in life sciences research laboratories often require chemicals and plasticware that are costly, toxic or pose a risk to the environment. Therefore, sustainable alternatives would be of interest, provided that they generate suitable data quality. Coomassie blue and silver staining are the most widely used methods for detecting proteins following electrophoresis in the laboratory. However, their use presents challenges in terms of safety and waste management. In the current study, aqueous extracts were prepared from a series of common food species and evaluated as alternative stains for protein detection. Beets, blueberries, purple cabbage, raspberries and strawberries were employed to stain identical proteins separated under the same conditions in electrophoresis gels. Extracts of the first two species resulted in protein bands that were detectable through visible light transillumination, whereas extracts from all five species generated specific protein bands under ultraviolet light. The raspberry-derived extract was selected for further study based on the brightness of the fluorescent protein bands and minimal background staining. For both bovine serum albumin and lysozyme at 2.5 μg and 0.5 μg protein per band, the mean signal intensities obtained with raspberry extract staining were just below half of those obtained with Coomassie blue. Furthermore, the mean intensities using raspberry extract were equivalent to those obtained using Coomassie blue in the detection of 0.1 μg protein. Therefore, raspberry could be used to produce an effective stain for the routine laboratory analysis of proteins
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