228 research outputs found
Multidimensional separation prior to mass spectrometry: Getting closer to the bottom of the iceberg
While prefractionation has previously been shown to improve results in MS
analysis, a novel combination provides an additional dimension of separation:
protein fractionation by SDS-PAGE followed by IEF of tryptic peptides before
separation by RP-LC [Atanassov and Urlaub, Proteomics 2013, 13, 2947-2955].
This three-step separation procedure prior to MS/MS substantially increases
proteome coverage and represents a further step toward a more comprehensive
analysis of complex proteomes
How shall we use the proteomics toolbox for biomarker discovery?
Biomarker discovery for clinical purposes is one of the major areas in which
proteomics is used. However, despite considerable effort, the successes have
been relatively scarce. In this perspective paper, we try to highlight and
analyze the main causes for this limited success, and to suggest alternate
strategies, which will avoid them, without eluding the foreseeable weak points
of these strategies. Two major strategies are analyzed, namely, the switch from
body fluids to cell and tissues for the initial biomarker discovery step or, if
body fluids must be analyzed, the implementation of highly selective protein
selection strategies
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
When 2D is not enough, go for an extra dimension
The use of an extra SDS separation in a different buffer system provide a
technique for deconvoluting 2D gel spots made of several proteins (Colignon et
al. Proteomics, 2013, 13, 2077-2082). This technique keeps the quantitative
analysis of the protein amounts and combines it with a strongly improved
identification process by mass spectrometry, removing identification
ambiguities in most cases. In some favorable cases, posttranslational variants
can be separated by this procedure. This versatile and easy to use technique is
anticipated to be a very valuable addition to the toolbox used in 2D gel-based
proteomics
Variations on a theme: Changes to electrophoretic separations that can make a difference
Electrophoretic separations of proteins are widely used in proteomic
analyses, and rely heavily on SDS electrophoresis. This mode of separation is
almost exclusively used when a single dimension separation is performed, and
generally represents the second dimension of two-dimensional separations.
Electrophoretic separations for proteomics use robust, well-established
protocols. However, many variations in almost all possible parameters have been
described in the literature over the years, and they may bring a decisive
advantage when the limits of the classical protocols are reached. The purpose
of this article is to review the most important of these variations, so that
the readers can be aware of how they can improve or tune protein separations
according to their needs. The chemical variations reviewed in this paper
encompass gel structure, buffer systems and detergents for SDS electrophoresis,
two-dimensional electrophoresis based on isoelectric focusing and
two-dimensional electrophoresis based on cationic zone electrophoresis
How to use 2D gel electrophoresis in plant proteomics
Two-dimensional electrophoresis has nurtured the birth of proteomics. It is
however no longer the exclusive setup used in proteomics, with the development
of shotgun proteomics techniques that appear more fancy and fashionable
nowadays.Nevertheless, 2D gel-based proteomics still has valuable features, and
sometimes unique ones, which make it often an attractive choice when a
proteomics strategy must be selected. These features are detailed in this
chapter, as is the rationale for selecting or not 2D gel-based proteomics as a
proteomic strategy
Keynotes on membrane proteomics
This review article deals with the specificities of the proteomics analysis
of membrane proteins
Silver Staining of Proteins in 2DE Gels
Silver staining detects proteins after electrophoretic separation on
polyacrylamide gels. Its main positive features are its excellent sensitivity
(in the low nanogram range) and the use of very simple and cheap equipment and
chemicals. The sequential phases of silver staining are protein fixation, then
sensitization, then silver impregnation, and finally image development. Several
variants of silver staining are described here, which can be completed in a
time range from 2 h to 1 day after the end of the electrophoretic separation.
Once completed, the stain is stable for several weeks
Detergents and Chaotropes for Protein Solubilization before Two-Dimensional Electrophoresis
Because of the outstanding separating capabilities of two-dimensional
electrophoresis for complete proteins, it would be advantageous to be able to
apply it to all types of proteins. Unfortunately, severe solubility problems
hamper the analysis of many classes of proteins, but especially membrane
proteins. These problems arise mainly in the extraction and isoelectric
focusing steps, and solutions are sought to improve protein solubility under
the conditions prevailing during isoelectric focusing. These solutions deal
mainly with chaotropes and new detergents, which are both able to enhance
protein solubility. The input of these compounds in proteomics analysis of
membrane proteins is discussed, as well as future directions.Comment: link to publisher's site http://biomed.humanapress.com
The proteomic to biology inference, a frequently overlooked concern in the interpretation of proteomic data: A plea for functional validation
Proteomics will celebrate its 20th year in 2014. In this relatively short
period of time, it has invaded most areas of biology and its use will probably
continue to spread in the future. These two decades have seen a considerable
increase in the speed and sensitivity of protein identification and
characterization, even from complex samples. Indeed, what was a challenge
twenty years ago is now little more than a daily routine. Although not
completely over, the technological challenge now makes room to another
challenge, which is the best possible appraisal and exploitation of proteomic
data to draw the best possible conclusions from a biological point of view. The
point developed in this paper is that proteomic data are almost always
fragmentary. This means in turn that although better than an mRNA level, a
protein level is often insufficient to draw a valid conclusion from a
biological point of view, especially in a world where PTMs play such an
important role. This means in turn that transformation of proteomic data into
biological data requires an important intermediate layer of functional
validation, i.e. not merely the confirmation of protein abundance changes by
other methods, but a functional appraisal of the biological consequences of the
protein level changes highlighted by the proteomic screens
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