We here report the construction of an E. coli expression system able to manufacture an unnatural amino acid by artificial biosynthesis. This can be orchestrated with incorporation into protein by amber stop codon suppression inside a living cell. In our case an alkyne-bearing pyrrolysine amino acid was biosynthesized and incorporated site-specifically allowing orthogonal double protein labeling.

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a β-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

Ten-eleven translocation hydroxylases (TET1-3) oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In neurons, increased 5hmC levels within gene bodies correlate positively with gene expression. The mechanisms controlling TET activity and 5hmC levels are poorly understood. In particular, it is not known how the neuronal TET3 isoform lacking a DNA-binding domain is targeted to the DNA. To identify factors binding to TET3, we screened for proteins that co-precipitate with TET3 from mouse retina and identified the transcriptional repressor REST as a highly enriched TET3-specific interactor. REST was able to enhance TET3 hydroxylase activity after co-expression and overexpression of TET3-activated transcription of REST target genes. Moreover, we found that TET3 also interacts with NSD3 and two other H3K36 methyltransferases and is able to induce H3K36 trimethylation. We propose a mechanism for transcriptional activation in neurons that involves REST-guided targeting of TET3 to the DNA for directed 5hmC generation and NSD3-mediated H3K36 trimethylation.

5-hydroxymethyluracil (hmU) is an oxidative DNA lesion and a potential intermediate in DNA repair–coupled DNA demethylation pathways. Isotopic labeling and MS reveal that hmU is produced directly by Tet-mediated oxidation of thymine in mouse embryonic stem cells, suggesting a functional role for hmU in stem cells.

MOTIVATION Mass spectrometry-based protein profiling has become a key technology in biomedical research and biomarker discovery. Sample preparation strategies that reduce the complexity of tryptic digests by immunoaffinity substantially increase throughput and sensitivity in proteomic mass spectrometry. The scarce availability of peptide-specific capture antibodies limits these approaches. Recently antibodies directed against short terminal motifs were found to enrich subsets of peptides with identical terminal sequences. This approach holds the promise of a significant gain in efficiency. TXP (Triple X Proteomics) and context-independent motif specific/global proteome survey binders are variants of this concept. Principally the binding motifs of such antibodies have to be elucidated after generating these antibodies. This entails a substantial effort in the lab, as it requires synthetic peptide libraries and numerous mass spectrometry experiments. RESULTS We present an algorithm for predicting the antibody-binding motif in a mass spectrum obtained from a tryptic digest of a common cell line after immunoprecipitation. The epitope prediction, based on peptide mass fingerprinting, reveals the most enriched terminal epitopes. The tool provides a P-value for each potential epitope, estimated by sampling random spectra from a peptide database. The second algorithm combines the predicted sequences to more complex binding motifs. A comparison with library screenings shows that the predictions made by the novel methods are reliable and reproducible indicators of the binding properties of an antibody.

The G protein-coupled receptor (GPCR) super-family comprises the largest and most diverse group of membrane receptors in eukaryotes. GPCRs are involved in a plethora of physiological functions in all kinds of tissues. Detailed knowledge about GPCR presence and expression levels in tissues can be very helpful for drug development as the majority of drugs are designed to modulate membrane receptors. Furthermore, it is known that many adverse drug effects result from GPCR interactions. However, very few satisfactory methods are currently available for the detection and quantification of GPCRs. The detection is complicated by their three-dimensional structure, their hydrophobic properties, and their localization in the plasma membrane with 7-trans-membrane domains and small cytosolic and extracellular domains. Due to these properties it is very difficult to generate specific antibodies directed against GPCRs for sandwich immunoassays and Western blot. We therefore designed an immunoaffinity- and mass spectrometry-based approach to analyze GPCR-specific signature peptides in tryptic digests in rat tissue lysates. The expression levels of four different GPCRs were determined using chemically labeled synthetic standard peptides. Here, we demonstrate for the first time, that peptide immunoaffinity MS-based methods can render a reliable and quantitative analysis of multi-membrane spanning receptor molecules.

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.

Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.