Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity

Gharahdaghi, Farzin; Weinberg, Catherine R.; Meagher, Denise A.; Imai, Brian S.; Mische, Sheenah M.

doi:10.1002/(sici)1522-2683(19990301)20:3<601::aid-elps601>3.0.co;2-6

Cited by 862 publications

(197 citation statements)

References 16 publications

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“…The procedure was similar for Coomassie-and silver-stained gel pieces, except that the latter were first decolorized by washing with 30 mL of 15 mM potassium ferricyanide/50 mM sodium thiosulfate [15], followed by three washes with 100 mL deionized water. Cysteine residues were reduced with DTT and derivatized by treatment with iodoacetamide.…”

Section: Preparation Of Samples For Mass Spectrometrymentioning

confidence: 99%

Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison

et al. 2001

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We have investigated the biological fluids--serum, cerebrospinal fluid, and urine--of three strains of rats; the present data extend our database (also available on-line) and may be of interest for pharmacological and toxicological investigation. Specifically, we have defined reference maps of the major protein components in cerebrospinal fluid and urine. Compartment-specific isoforms were recognized for transferrin and transthyretin. Mass spectrometric data established the cleavage site of the signal peptide and identified the N-terminal blocking group of prostaglandin D synthase from rat cerebrospinal fluid. A previously undescribed member of the family of low molecular mass rat urinary proteins was characterized as containing a sequence similar, but not identical, to the N-terminal region of rat urinary protein-2 (RUP-2), and divergent from RUP-1.

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Section: Preparation Of Samples For Mass Spectrometrymentioning

confidence: 99%

Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison

et al. 2001

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“…At this concentration the band was barely visible by silver staining, but all 13 residues could easily be deduced from the MS 2 spectrum, with mass errors of only 0.01% [120]. Other reported LODs are in the same range: 20 ng of protein were detected after destaining, digestion and MALDI-TOF-MS [85]. An average LOD of 100-500 fmol for 2-DE and subsequent MALDI-MS has been estimated in [37]; 20-200 fmol were achieved in [41]; an average LOD of 1-1.5 pmol has been estimated for 2-DE, subsequent peptide sequencing and ESI-MS analysis [37].…”

Section: Detectionmentioning

confidence: 99%

Possibilities to improve automation, speed and precision of proteome analysis: A comparison of two-dimensional electrophoresis and alternatives

2001

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Proteome analysis requires fast methods with high separation efficiencies in order to screen the various cell and tissue types for their proteome expression and monitor the effect of environmental conditions and time on this expression. The established two-dimensional gel electrophoresis (2-DE) is by far too slow for a consequential screening. Moreover, it is not precise enough to observe changes in protein concentrations. There are various approaches that promise faster, automated proteome analysis. This article concentrates on capillary (CT isoelectric focusing coupled to mass spectrometry (CIEF-MSn) and preparative IEF followed by size-exclusion chromatography, hyphenated with MS (PIEF-SEC-MS). These two approaches provide a similar separation pattern as the established 2-DE technique and therefore allow for the continued use of data based on this traditional approach. Their performances have been discussed and compared to 2-DE, evaluating 169 recent articles. Data on analysis time, automation, the detection limit, quantitation, peak capacity, mass and pI accuracy, as well as on the required sample amount are compared in a table.

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“…Gels were prepared for digestion by staining with special silver stain, cutting the spots of interest out with a knife, and placing them in separate tubes where they were destained according to Gharahdaghi et al [14]. The samples were then dehydrated with 200 mL acetonitrile, the acetonitrile removed and the sample dried in a SpeedVac concentrator.…”

Section: In-gel Digestion and Maldi Analysis Of Special Silver-stainementioning

confidence: 99%

A proteomic approach to rapidly elucidate oligodendrocyte-associated proteins expressed in the myelinating rat optic nerve

et al. 2002

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The lack of cDNA libraries derived from myelinating oligodendrocytes has hampered attempts to identify proteins associated with myelination during normal development or with remyelination after insult or disease. We, therefore, established a new method to elucidate such proteins by coupling the techniques of X-irradiation, two-dimensional (2-D) gel electrophoresis, and mass spectrometry. Specifically, 2-D gel protein profiles of normal optic nerves were compared with those of X-irradiated rat optic nerves, which were absent of oligodendrocytes, to elucidate oligodendrocyte-associated proteins. The subsequent identification of these oligodendrocyte-associated proteins was accomplished by mass spectrometry. The results presented in this paper demonstrate the potential of the X-irradiated optic nerve model system combined with proteomic techniques to rapidly elucidate oligodendrocyte-associated proteins expressed in vivo.

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Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity

Cited by 862 publications

References 16 publications

Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison

Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison

Possibilities to improve automation, speed and precision of proteome analysis: A comparison of two-dimensional electrophoresis and alternatives

A proteomic approach to rapidly elucidate oligodendrocyte-associated proteins expressed in the myelinating rat optic nerve

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