Improved mass spectrometry compatibility is afforded by ammoniacal silver staining

Autor: Mireille Chevallet, Thierry Rabilloud, Emmanuelle Leize-Wagner, Hélène Diemer, Alain Van Dorsselaer, Sylvie Luche
Přispěvatelé: Contrôle moléculaire de la réponse immune specifique, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pluridisciplinaire Hubert Curien (IPHC), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS)
Rok vydání: 2006
Předmět:
Proteomics
Silver Staining
Low protein
Mass spectrometry
01 natural sciences
Biochemistry
MESH: Silver Staining
Mass Spectrometry
Article
Adduct
Silver stain
03 medical and health sciences
chemistry.chemical_compound
Ammonia
[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry
Molecular Biology/Genomics [q-bio.GN]
Humans
Quantitative Biology - Genomics
Electrophoresis
Gel
Two-Dimensional
MESH: Ammonia
Molecular Biology
030304 developmental biology
Genomics (q-bio.GN)
0303 health sciences
MESH: Humans
Chromatography
MESH: Peptides
Chemistry
MESH: Proteomics
010401 analytical chemistry
Reproducibility of Results
MESH: Electrophoresis
Gel
Two-Dimensional
0104 chemical sciences
3. Good health
Staining
MESH: Reproducibility of Results
Silver nitrate
FOS: Biological sciences
electrophoresis
MESH: Isoelectric Focusing
Mass spectrum
Isoelectric Focusing
Peptides
MESH: Spectrum Analysis
Mass
Zdroj: Proteomics
Proteomics, 2006, 6 (8), pp.2350-4. ⟨10.1002/pmic.200500567⟩
Proteomics, Wiley-VCH Verlag, 2006, 6 (8), pp.2350-4. ⟨10.1002/pmic.200500567⟩
ISSN: 1615-9861
1615-9853
DOI: 10.1002/pmic.200500567
Popis: 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.com
Databáze: OpenAIRE
Externí odkaz: