2,169 research outputs found
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
Sweet silver: A formaldehyde-free silver staining using aldoses as developing agents, with enhanced compatibility with mass spectrometry
Protein detection methods after electrophoresis have to be sensitive,
homogeneous, and not to impair downstream analysis of proteins by MS. Speed,
low cost, and user friendliness are also favored features. Silver staining
combines many of these features, but its compatibility with MS is limited. We
describe here, a new variant of silver staining that is completely
formaldehyde-free. Reducing sugars in alkaline borate buffer are used as
developers. While keeping the benefits of silver staining, this method is shown
to afford a much better performance in terms of compatibility with MS, both in
PMF by MALDI and in LC/ESI/MS/MS
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
Alterations of the mitochondrial proteome caused by the absence of mitochondrial DNA: A proteomic view
The proper functioning of mitochondria requires that both the mitochondrial
and the nuclear genome are functional. To investigate the importance of the
mitochondrial genome, which encodes only 13 subunits of the respiratory
complexes, the mitochondrial rRNAs and a few tRNAs, we performed a comparative
study on the 143B cell line and on its Rho-0 counterpart, i.e., devoid of
mitochondrial DNA. Quantitative differences were found, of course in the
respiratory complexes subunits, but also in the mitochondrial translation
apparatus, mainly mitochondrial ribosomal proteins, and in the ion and protein
import system, i.e., including membrane proteins. Various mitochondrial
metabolic processes were also altered, especially electron transfer proteins
and some dehydrogenases, but quite often on a few proteins for each pathway.
This study also showed variations in some hypothetical or poorly characterized
proteins, suggesting a mitochondrial localization for these proteins. Examples
include a stomatin-like protein and a protein sharing homologies with bacterial
proteins implicated in tyrosine catabolism. Proteins involved in apoptosis
control are also found modulated in Rho-0 mitochondria.Comment: website publisher: http://www3.interscience.wiley.com
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 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
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)
The gene-centric human proteome project: organisation, scope and limitations
Comunicaciones a congreso
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
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