Identification of tissue proteins by amino acid analysis after purification by two-dimensional electrophoresis

Jungblut, Peter R.; Dzionara, M.; Klose, Joachim; Wittmann-Leibold, B.

doi:10.1007/bf01024960

Cited by 48 publications

(18 citation statements)

References 28 publications

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“…1). The N-terminally blocked protein was first identified by amino acid analysis [6] and search in the National Biomedical Research Foundation (NBRF) sequence database by the search program ASA [2]. This program uses a standard deviation S between the determined amino acid composition and that of the protein in comparison with the database as a measure for the probability of identity.…”

Section: Resultsmentioning

confidence: 99%

“…About one-tenth of these proteins can be identified by protein sequencing using automated Edman degradation with a sensitivity in the lower picomole range. Amino acid analysis may be an additional help for identification in the same sensitivity range [2]. A higher sensitivity has been obtained using immunoblotting [3], but a prior hypothesis of the identity of the protein and highly specific antibodies are necessary for this strategy of identification.…”

Section: Introductionmentioning

confidence: 98%

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Identification of human myocardial proteins separated by two‐dimensional electrophoresis with matrix‐assisted laser desorption/ionization mass spectrometry

Thiede¹,

Otto²,

Zimny‐Arndt³

et al. 1996

Electrophoresis

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Disease-associated proteins separated by two-dimensional electrophoresis (2-DE) are often in the femtomole range. Identification of 2-DE separated proteins by sequencing and amino acid analysis is limited to the lower picomole range. Identification down to the femtomole range can be achieved by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). We optimized the measurement by MALDI-MS for the analysis of proteolytic digests of 2-DE-separated proteins. The direct analysis of peptide mixtures can be used for rapid and sensitive protein identification. In some cases, more information about the protein can be obtained by separating the peptides by micro high-performance liquid chromatography (HPLC) before employing MALDI-MS analysis. More peptides are found than in the mixtures, and comparison of HPLC patterns can reveal some differences to be post-translational modifications of proteins, even in the case of identical peptide mass fingerprints. Furthermore, carboxy-terminal sequencing by on-target carboxypeptidase P digestion can be used to confirm the obtained result without the need for more material. The search program FRAGFIT was modified and renamed FRAGMOD to include the modifications of methionine and tryptophan oxidation and alkylation of cysteine by acrylamide into the mass search. By applying this procedure, 15 proteins were identified, among them two different putative phosphorylated forms of two proteins, a putative N-terminal blocking group and four dilated cardiomyopathy-associated proteins. The resulting approach for the identification may be used for large-scale investigations of 2-DE-separated proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Identification of human myocardial proteins separated by two‐dimensional electrophoresis with matrix‐assisted laser desorption/ionization mass spectrometry

Thiede¹,

Otto²,

Zimny‐Arndt³

et al. 1996

Electrophoresis

View full text Add to dashboard Cite

show abstract

“…Quantification of Differences in Protein Abundance between M. tuberculosis H37Rv and M. bovis BCG-Absolute quantification of proteins is a difficult task and at best achieved by amino acid composition analysis (24). Therefore, most proteomic experiments attempt to obtain relative quantitative information by determining the abundance ratios of proteins present in two samples.…”

Section: Fig 3 Comparison Of Proteins Identified By the Icat-lc/ms mentioning

confidence: 99%

Complementary Analysis of the Mycobacterium tuberculosis Proteome by Two-dimensional Electrophoresis and Isotope-coded Affinity Tag Technology

Schmidt

Donahoe

Hagens

et al. 2004

Molecular & Cellular Proteomics

169

138

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Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M r proteins and was complemented by the 2-DE/MS method, which showed a preference for low M r proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied. with mass spectrometry (MS) and have revealed about 1,800 distinct spots separated by 2-DE (1). About 350 of these were identified, and the comparison of the protein patterns of virulent and attenuated strains identified several proteins that are being studied further as potential vaccine candidates (1, 2). More than two million deaths/year and eight million new infections are caused by M. tuberculosis, the bacterium responsible for tuberculosis (3). Genomics, transcriptomics, and proteomics, which form the rationale basis to develop new therapeutic and preventive strategies, have been applied to combat this disease. The complete genome of M. tuberculosis comprises about 4,000 genes, which were classified in six protein classes and 30 subclasses (4).The smallest unit of the proteome, the protein species, is defined by its chemical structure (5). Therefore, each modification of a protein leads to a new protein species. These are successfully resolved by 2-DE if they differ by at least one charge or by at least several hundred daltons in mass. Quantification by 2-DE requires a high degree of pattern reproducibility, which is difficult to achieve in a multistep and parallel procedure.To alleviate limi...

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“…For analytical investigations, proteins were detected by silver staining in 0.75 mm gels, while 1.5 mm gels were used for preparative purposes. Proteins were directly stained with Coomassie Brilliant Blue R-250, or they were stained after blotting [18] either with sulforhodamine B (Sigma, Deisenhofen, Germany) [15] or Coomassie Brilliant Blue R-250 [19].…”

Section: Sample Preparation and 2-dementioning

confidence: 99%

High‐performance human myocardial two‐dimensional electrophoresis database: Edition 1996

et al. 1996

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The master gel of the human myocardial two-dimensional electrophoresis (2-DE) gel database contains about 3300 protein spots characterized in terms of isoelectric point (pI) and molecular mass. A high-performance technique was applied, using large gels (23 x 30 cm). Isoelectric focusing with anodic sample preparation and nonequilibrium running conditions (NEPHGE) was combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 15% acrylamide gels in the second dimension. The range of pI extends from pH 4.5 to 9.6. Seventy proteins were identified by combinations of amino acid analysis, N-terminal and internal sequencing, immunostaining, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting, post-source decay MALDI-MS and ladder sequencing by carboxypeptidase P. The identification of additional proteins, not found in the master gel, was achieved by immunoblotting. Unequivocal identification with high sensitivity and good yield was obtained by combining internal sequencing and MALDI-MS. In-gel digestion, the concentration and purification of peptides in a peptide collecting device, and the improved FRAGMOD program for peptide mass fingerprinting have added to the security and sensitivity of identification. The high-performance human myocardial 2-DE database was built up with proteins detected by the TOPSPOT program. Spots within six sections of the whole pattern are clickable. Protein description includes detailed information about identification, characterization, and links to the related SWISS-PROT, other 2-DE databases and Medline entries. The database is constructed in accordance with four of the rules for a federated database.

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Identification of tissue proteins by amino acid analysis after purification by two-dimensional electrophoresis

Cited by 48 publications

References 28 publications

Identification of human myocardial proteins separated by two‐dimensional electrophoresis with matrix‐assisted laser desorption/ionization mass spectrometry

Identification of human myocardial proteins separated by two‐dimensional electrophoresis with matrix‐assisted laser desorption/ionization mass spectrometry

Complementary Analysis of the Mycobacterium tuberculosis Proteome by Two-dimensional Electrophoresis and Isotope-coded Affinity Tag Technology

High‐performance human myocardial two‐dimensional electrophoresis database: Edition 1996

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