Functional proteomics of signal transduction by membrane receptors

Zimmermann, Jasminka Godovac; Šoškić, Vukic; Poznanović, Slobodan; Brianza, Federico

doi:10.1002/(sici)1522-2683(19990101)20:4/5<952::aid-elps952>3.0.co;2-a

Cited by 59 publications

(28 citation statements)

References 34 publications

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“…Immunochemicals specific for P-tyrosine, P-serine and P-threonine have been developed as much as for the P-variants of individual proteins. The former should allow surveying the whole phosphoproteome [134,135], the latter assessing the ratio between/among phosphorylation isoforms; some aspecific binding of the antibodies may be a confounding factor in these investigations. Sample enrichment in phosphoproteins using immunological (anti-phosphoserine immobilized onto agarose [136]) or affinity (Fe 31 -laden IMAC resin [137], metal oxide/hydroxide affinity chromatography [138]) procedures may be a valuable alternative.…”

Section: Phosphoproteinsmentioning

confidence: 99%

Protein stains for proteomic applications:Which, when, why?

Crawford²,

2006

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This review recollects literature data on sensitivity and dynamic range for the most commonly used colorimetric and fluorescent dyes for general protein staining, and summarizes procedures for the most common PTM-specific detection methods. It also compiles some important points to be considered in imaging and evaluation. In addition to theoretical considerations, examples are provided to illustrate differential staining of specific proteins with different detection methods. This includes a large body of original data on the comparative evaluation of several pre-and post-electrophoresis stains used in parallel on a single specimen, horse serum run in 2-DE (IPG-DALT). A number of proteins/protein spots are found to be over-or under-revealed with some of the staining procedures.

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Section: Phosphoproteinsmentioning

confidence: 99%

Protein stains for proteomic applications:Which, when, why?

Crawford²,

2006

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“…In the transformation process changes in protein expression may result where proteins are expressed at elevated or reduced levels [17][18][19][20][21][22][23][24][25][26]. Alternatively, important mechanisms in the cancer process often involve structural changes such as sequence variations or post-translational modifications [26][27][28][29][30][31][32]. As a cell transforms from a normal to a malignant cell, such changes in protein expression ultimately reflect themselves in the phenotype of the cell.…”

Section: Introductionmentioning

confidence: 99%

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique

Wang¹,

Kachman²,

Schwartz³

et al. 2002

Electrophoresis

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A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two-dimensional (2-D) liquid separations/mass mapping technique. This method uses a 2-D liquid separation of proteins from whole cell lysates coupled on-line to an electrospray ionization-time of flight (ESI-TOF) mass spectrometer to map the accurate intact molecular weight (M(r)) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase high-performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI-TOF-MS provides an image of pI versus M(r) analogous to 2-D gel electrophoresis. Each protein is then identified based upon matrix-assisted laser desorption/ionization (MALDI)-TOF-MS peptide mapping and intact M(r) so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact M(r) together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 M(r) bands are detected over 17 pI fractions from pH 4 to 12 and a M(r) range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein M(r) is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein M(r) map may serve as a highly reproducible standard Web-based method for comparing proteins from related human cell lines.

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“…However, this approach cannot discriminate between different classes of phosphorylation sites and is relatively insensitive. Recent progress in two-dimensional gel electrophoresis followed by mass spectrometry allows the comprehensive analysis of the tyrosine phosphorylation state in complex mixtures of proteins (13)(14)(15). However, this approach has the major disadvantage that relatively large amounts of protein are needed.…”

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confidence: 99%

Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding

Nollau

Mayer

2001

Proc. Natl. Acad. Sci. U.S.A.

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Reversible tyrosine phosphorylation plays a crucial role in signal transduction, regulating many biological functions including proliferation, differentiation, and motility. The comprehensive characterization of the tyrosine phosphorylation state of a cell is of great interest for understanding the mechanisms that underlie signaling; however, current methods for analyzing tyrosinephosphorylated proteins in crude protein extracts provide limited information, or are laborious and require relatively large amounts of protein. We have developed a simple, rapid, and flexible competitive binding assay based on the far-Western blot technique, in which a battery of Src homology 2 domain probes is used to detect patterns of specific tyrosine-phosphorylated sites. We demonstrate that distinct profiles of tyrosine phosphorylation can be detected with high sensitivity and specificity and low background. This proteomic approach can be used to rapidly profile the global tyrosine phosphorylation state of any cell of interest and has obvious applications as a molecular diagnostic tool, for example in the classification of tumors. The general strategy we describe here is not limited to Src homology 2 domains and could be used to profile the binding sites for any class of protein interaction domain.T he process of signal transduction is essential for cellular functions such as proliferation, differentiation, cytoskeletal organization, and cell survival, and aberrant signaling is a hallmark of many diseases (1-3). Specific protein-protein interactions play a key role in this process by mediating the assembly of complexes or relocalization of proteins in response to signals (4). Reversible tyrosine phosphorylation is an early step in the transduction of many types of signals in multicellular organisms. Signaling proteins phosphorylated on tyrosine residues are specifically recognized by Src Homology 2 (SH2) modular protein interaction domains; thus, creation of SH2 binding sites serves to transmit a signal by altering the local concentrations of proteins containing SH2 domains and bringing them into contact with new partners or substrates (5, 6). One way in which we can define the signaling state of the cell, therefore, is by the presence or absence of binding sites for various SH2 domains, the subcellular localization of those sites, and the ensemble of proteins that are available to bind to those sites. SH2 domains, which are found in a wide variety of signaling proteins, consist of Ϸ100 aa and can be separated from the original protein without loss of function (7). Their ligand binding surfaces specifically interact with phosphotyrosine (PTyr) in the context of short linear sequence motifs. The specificity of the interaction is determined by the amino acid composition of the core binding site, with the general motif pYxx⌿ (where pY stands for PTyr, ⌿ for hydrophobic amino acids, and x for selected amino acids important for specific interaction) (8, 9). The affinities for SH2 domain-ligand interactions are moderate, in the range o...

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Functional proteomics of signal transduction by membrane receptors

Cited by 59 publications

References 34 publications

Protein stains for proteomic applications:Which, when, why?

Protein stains for proteomic applications:Which, when, why?

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique

Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding

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