Functionalized membrane supports for covalent protein microsequence analysis

Coull, James; Pappin, Darryl; Mark, Jonathan; Aebersold, Ruedi; Köster, Hubert

doi:10.1016/0003-2697(91)90157-o

Cited by 60 publications

(17 citation statements)

References 18 publications

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“…Protein samples were separated by SDS-PAGE in 12.5% (m v -1 ) polyacrylamide gels (Laemmli, 1970) and stained with Coomassie Brilliant Blue R-250 or electro-blotted onto a PVDF membrane (Sequi-Blot TM PVDF Membrane, Trans-Blot® SD, Semi-dry transfer cell, Bio-Rad, Hercules, USA) as described (Coull et al, 1991;Matsudaira, 1987). The protein of interest was excised from the stained membrane and sequenced Nterminally using a gas phase sequencer (Procise TM 491 Protein Sequencer, 11 785 Programmable Absorbance Detector, 140 C Microgradient System, Applied Biosystems, Weiterstadt, Germany).…”

Section: Sds-page Blotting and N-terminal Sequencing Of Proteinsmentioning

confidence: 99%

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

Oelschlaeger

Lange

Schmitt

et al. 2003

Appl Microbiol Biotechnol

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In order to produce the atrazine-specific scFv K411B, it was expressed in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the scFv was N-terminally fused to the pelB leader, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly: The expression level of the scFv with leader was 2.3 times higher than that of the protein without leader. To further investigate this, the respective translation profiles were generated by coupled in vitro transcription/translation assays and gave according results. Periplasmic expression resulted in only 10% correctly folded scFv. The same percentage was obtained when the scFv was expressed in vitro, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of the scFv with EGFP fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.3

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Section: Sds-page Blotting and N-terminal Sequencing Of Proteinsmentioning

confidence: 99%

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

Oelschlaeger

Lange

Schmitt

et al. 2003

Appl Microbiol Biotechnol

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“…32P-labelled phosphotryptic peptides were scraped from TLC plates and eluted as described above. The peptide was applied to Sequelon acrylamine membrane (Milligen), dried, and coupled (10). After four washes with 9% trifluoroacetic acid-27% acetonitrile (55), the membranebound peptide was placed into an Applied Biosystems 470A sequencer.…”

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confidence: 99%

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

Schaller¹,

Hildebrand²,

Shannon³

et al. 1994

Mol. Cell. Biol.

1,068

946

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The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp6Orc and pp590 ', via ing sequence motif (43, 44). The specificity of such binding is dictated by the residues flanking the site of tyrosine phosphorylation as well as the structural characteristics of a particular SH2 domain (12,54). Examples drawn from growth factor receptor PTK signalling illustrate the importance of SH2-phosphotyrosine interactions in the transmission of cytoplasmic signals. For example, receptor PTK autophosphorylation can recruit SH2-containing substrates to the receptor complex, thereby facilitating their phosphorylation. In turn, the phosphorylation of certain substrates appears to be critical for the regulation of their activities (for example, the growth factordependent activation of phospholipase C&y [13,23,40]). SH2-phosphotyrosine interactions also provide a mechanism by which cytosolic enzymes whose substrates are present in cell membranes can be translocated to the proximity of their substrates (for example, phospholipase C&y and phosphatidylinositol 3-kinase, cytosolic enzymes whose substrates are membrane lipids [9,26,37], and Sos, a cytosolic protein that functions as a guanine nucleotide exchange factor for the membrane-associate protein p2lras [34]). Binding of a tyrosinephosphorylated protein to an SH2 domain-containing enzyme can lead directly to an increase in enzymatic activity. For example, binding of a tyrosine-phosphorylated peptide to the SH2 domain of the regulatory 85-kDa subunit of phosphatidylinositol 3-kinase (P13K) induces conformational changes in p85 and the concomitant increase in enzymatic activity of the holoenzyme (2,6,35,41). Finally, SH2-phosphotyrosine interactions may be critical for the negative regulation of enzymatic activity. For example, c-Src kinase activity is regulated by the binding of its SH2 domain to the C-terminal regulatory site of tyrosine phosphorylation, 8). Thus, phosphotyrosine-SH2 interactions not only mediate protein-protein complex formation but are also intimately involved in the regulation of the activity of a number of signalling molecules.The PTK pp125FAK is phosphorylated on tyrosine in response to the engagement of cell surface integrins with com-1680 on April 2, 2019 by guest

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“…Protease digestion of the protein followed by HPLC separation of the peptide mixture and collection of the radioactive fractions can be coupled with Edman sequencing to determine the site of phosphorylation (5)(6)(7)(8). However, Edman sequencing has certain limitations (9,10) that have led to the rise of mass spectrometry as the preferred tool for peptide sequencing.…”

mentioning

confidence: 99%

Automated Identification and Quantification of Protein Phosphorylation Sites by LC/MS on a Hybrid Triple Quadrupole Linear Ion Trap Mass Spectrometer

Williamson¹,

Marchese²,

Morrice

2006

Molecular & Cellular Proteomics

108

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Complete phosphorylation mapping of protein kinases was successfully undertaken using an automated LC/ MS/MS approach. This method uses the direct combination of triple quadrupole and ion trapping capabilities in a hybrid triple quadrupole linear ion trap to selectively identify and sequence phosphorylated peptides. In particular, the use of a precursor ion scan of m/z ؊79 in negative ion mode followed by an ion trap high resolution scan (an enhanced resolution scan) and a high sensitivity MS/MS scan (enhanced product ion scan) in positive mode is a very effective method for identifying phosphorylation sites in proteins at low femtomole levels. Coupling of this methodology with a stable isotope N-terminal labeling strategy using iTRAQ TM reagents enabled phosphorylation mapping and relative protein phosphorylation levels to be determined between the active and inactive forms of the protein kinase MAPKAPK-1 in the same LC/MS run. Reversible protein phosphorylation is one of the most important post-translational modifications (PTMs) 1 with as much as one-third of all proteins in a mammalian cell being phosphorylated (1). Phosphate introduction occurs through a large group of protein kinases that are estimated to constitute between 2-5% of the expressed genome (2-4). Protein phosphorylation mediates most of the signal transduction pathways in eukaryotic cells, controls cell processes such as metabolism and transcription, and plays a role in intercellular communication. It is important to determine the exact site of protein phosphorylation to elucidate the physiological role of a particular phosphorylation event. Once phosphorylation sites are identified, it is extremely valuable to determine how these protein phosphorylation sites change in response to extracellular stimuli or intracellular events. Studying phosphorylation is very challenging in a highly complex protease digest mixture as individual phosphorylation sites are often only partially phosphorylated and therefore at very low concentrations.Traditional ways to localize phosphorylation sites involve the use of 32 P to label the proteins under investigation. Protease digestion of the protein followed by HPLC separation of the peptide mixture and collection of the radioactive fractions can be coupled with Edman sequencing to determine the site of phosphorylation (5-8). However, Edman sequencing has certain limitations (9, 10) that have led to the rise of mass spectrometry as the preferred tool for peptide sequencing.The ability of mass spectrometry to detect and characterize sites of phosphorylation is not without its problems. Signals of phosphopeptides in the MS spectra are, in most cases, suppressed by the relatively high concentration of non-phosphopeptides present in proteolytic digests of these samples. Even LC/MS of a single protein digest can result in phosphopeptides going undetected due to suppression from coeluting, more basic peptides. Therefore, a better approach to selectively identify and characterize phosphopeptides from digest mixtures is hi...

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Functionalized membrane supports for covalent protein microsequence analysis

Cited by 60 publications

References 18 publications

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

Automated Identification and Quantification of Protein Phosphorylation Sites by LC/MS on a Hybrid Triple Quadrupole Linear Ion Trap Mass Spectrometer

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