7 research outputs found
Die Talsperre. 6. Jahrgang [1907/08], Nummer 31 : Zeitschrift für Wasserwirtschaft, Wasserrecht, Meliorationswesen und allgemeine Landeskultur
Additional file 5. RNA-seq analysis. List of 727 sequencing runs searched with a DEspR query sequence in the NCBI Sequence Read Archive
MOESM1 of Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein
Additional file 1: Figure S1. NCBI designation of Dear (alias DEspR) gene as pseudogene—updated May 2016
Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli
Expansion
of the genetic code through engineering the translation
machinery has greatly increased the chemical repertoire of the proteome.
This has been accomplished mainly by read-through of UAG or UGA stop
codons by the noncanonical aminoacyl-tRNA of choice. While stop codon
read-through involves competition with the translation release factors,
sense codon reassignment entails competition with a large pool of
endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase
to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number
of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements
of amino acid incorporation yields carried out in a selected reaction
monitoring experiment revealed that the 3-I-Phe abundance at the Ser<sub>208</sub>AGU codon in superfolder GFP was 65 ± 17%. This method
also allowed quantification of other amino acids (serine, 33 ±
17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete
with 3-I-Phe at both the aminoacylation and decoding steps of translation
for incorporation at the same codon position. Reassignments of different
serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching
tRNA<sup>Pyl</sup> anticodon variants were met with varying success,
and our findings provide a guideline for the choice of sense codons
to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA
synthetase (IFRS)/tRNA<sup>Pyl</sup> pair can efficiently outcompete
the cellular machinery to reassign select sense codons in wild-type E. coli
Impact of Detergents on Membrane Protein Complex Isolation
Detergents
play an essential role during the isolation of membrane
protein complexes. Inappropriate use of detergents may affect the
native fold of the membrane proteins, their binding to antibodies,
or their interaction with partner proteins. Here we used cadherin-11
(Cad11) as an example to examine the impact of detergents on membrane
protein complex isolation. We found that mAb 1A5 could immunoprecipitate
Cad11 when membranes were solubilized by dodecyl maltoside (DDM) but
not by octylglucoside, suggesting that octylglucoside interferes with
Cad11–mAb 1A5 interaction. Furthermore, we compared the effects
of Brij-35, Triton X-100, cholate, CHAPSO, Zwittergent 3-12, Deoxy
BIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation.
We found that all detergents except Brij-35 could solubilize Cad11
from the membrane. Upon immunoprecipitation, we found that β-catenin,
a known cadherin-interacting protein, was present in Cad11 immune
complex among the detergents tested except Brij-35. However, the association
of p120 catenin with Cad11 varied depending on the detergents used.
Using isobaric tag for relative and absolute quantitation (iTRAQ)
to determine the relative levels of proteins in Cad11 immune complexes,
we found that DDM and Triton X-100 were more efficient than cholate
in solubilization and immunoprecipitation of Cad11 and resulted in
the identification of both canonical and new candidate Cad11-interacting
proteins
Cross-laboratory quantification of BSA-BDP cross-linked peptide pairs.
<p><b>A.</b> Average cross-linked peptide response curve comparing data collected in the Bruce Lab and CSHL for 30 cross-linked peptide pairs. <b>B.</b> Scatter plot illustrating the R<sup>2</sup> values for linear regression analysis of the data shown in A and B.</p
Quantification of BSA cross-linked peptide pairs with Skyline.
<p><b>A.</b> MS2 spectrum for the cross-linked peptide pair linking residues K235-K28 (ALK<sup>235</sup>AWSVAR_DTHK<sup>28</sup>SEIAHR), obtained from a 500 ng injection of cross-linked BSA digest. <b>B.</b> Extracted ion chromatograms for the PRM transitions observed for the cross-linked peptide pair in A. <b>C.</b> Skyline generated bar plot illustrating the normalized peak areas for the cross-linked peptide pair linking K28-K235. Peak areas are shown for triplicate analyses of varying injection amounts (100, 200, 500, and 1000 ng cross-linked BSA digest). Bars are color coded to indicate the contribution of each individual transition to the total peak area and match the color scheme in panel B. </p
Experimental outline.
<p><b>A.</b> Biological samples are prepared for qXL-MS comparing two or more conditions. The samples are treated with chemical cross-linker either as (1) a mixed sample if SILAC labeling was used or (2) as separate samples if carrying out a label free experiment or using isotopically labeled cross-linkers. Following the cross-linking reaction proteins are extracted, enzymatically digested, and subjected to various strategies (i.e. strong cation exchange and affinity chromatography) for enrichment cross-linked peptide pairs. <b>B.</b> LC-MS analysis of samples enriched for cross-linked peptide pairs is carried out. This consists of reversed phase chromatographic separation by LC followed by analysis by MS. The mass spectrometer is operated in PRM mode where an inclusion list of <i>m/z</i> values for the precursor ions of interest is used to target specific cross-linked peptides. The PRM mass spectrometric analysis used here consists of three steps including isolation of precursor ions, fragmentation by collision with neutral gasses, and detection of mass to charge ratios of the resulting fragment ions. <b>C)</b> Resulting MS2 data are converted into transition lists and imported into Skyline for analysis.</p