Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for twostep purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 M urea, by Ni 2؉ chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein com- Mass spectrometric analysis of proteins has tremendously contributed to our understanding of biological systems. Mapping of covalent protein modifications by mass spectrometric approaches has made it possible to identify and rapidly evaluate the biological significance of modifications. In addition, identification of protein complexes by mass spectrometry has allowed investigators to connect cellular pathways and to describe the dynamics of protein complexes (1, 2). These approaches typically require a high degree of purification of proteins or protein complexes. Importantly to get a genuine picture of the in vivo situation it is essential to avoid any changes in protein modification or protein complex composition that might occur during the purification procedure. Two-step purification strategies have been proven to be very effective in reducing nonspecific background, which is particularly important for the analyses of complex protein samples (3). The first widely and successfully used tandem affinity tag was the TAP 1 tag, which consists of the immunoglobulin-interacting domain of Protein A and a calmodulinbinding peptide (CBP) a...

We developed an integrated proteomic approach to decipher in vivo protein-protein interactions and applied this strategy to globally map the 26 S proteasome interaction network in yeast. We termed this approach QTAX for quantitative analysis of tandem affinity purified in vivo cross-linked (X) protein complexes. For this work, in vivo formaldehyde cross-linking was used to freeze both stable and transient interactions occurring in intact cells prior to lysis. To isolate cross-linked protein complexes with high purification efficiency under fully denaturing conditions, a new tandem affinity tag consisting of a hexahistidine sequence and an in vivo biotinylation signal was adopted for affinity-based purification. Tandem affinity purification after in vivo cross-linking was combined with tandem mass spectrometry coupled with a quantitative SILAC (stable isotope labeling of amino acids in cell culture) strategy to carry out unambiguous protein identification and quantification of specific protein interactions.

Cloning of whole genomes of the genus Mycoplasma in yeast has been an essential step for the creation of the first synthetic cell. The genome of the synthetic cell is based on Mycoplasma mycoides, which deviates from the universal genetic code by encoding tryptophan rather than the UGA stop codon. The feature was thought to be important because bacterial genes might be toxic to the host yeast cell if driven by a cryptic promoter active in yeast. As we move to expand the range of bacterial genomes cloned in yeast, we extended this technology to bacteria that use the universal genetic code. Here we report cloning of the Acholeplasma laidlawii PG-8A genome, which uses the universal genetic code. We discovered that only one A. laidlawii gene, a surface anchored extracellular endonuclease, was toxic when cloned in yeast. This gene was inactivated in order to clone and stably maintain the A. laidlawii genome as a centromeric plasmid in the yeast cell.

BackgroundSynthetic genomic approaches offer unique opportunities to use powerful yeast and Escherichia coli genetic systems to assemble and modify chromosome-sized molecules before returning the modified DNA to the target host. For example, the entire 1 Mb Mycoplasma mycoides chromosome can be stably maintained and manipulated in yeast before being transplanted back into recipient cells. We have previously demonstrated that cloning in yeast of large (> ~ 150 kb), high G + C (55%) prokaryotic DNA fragments was improved by addition of yeast replication origins every ~100 kb. Conversely, low G + C DNA is stable (up to at least 1.8 Mb) without adding supplemental yeast origins. It has not been previously tested whether addition of yeast replication origins similarly improves the yeast-based cloning of large (>150 kb) eukaryotic DNA with moderate G + C content. The model diatom Phaeodactylum tricornutum has an average G + C content of 48% and a 27.4 Mb genome sequence that has been assembled into chromosome-sized scaffolds making it an ideal test case for assembly and maintenance of eukaryotic chromosomes in yeast.ResultsWe present a modified chromosome assembly technique in which eukaryotic chromosomes as large as ~500 kb can be assembled from cloned ~100 kb fragments. We used this technique to clone fragments spanning P. tricornutum chromosomes 25 and 26 and to assemble these fragments into single, chromosome-sized molecules. We found that addition of yeast replication origins improved the cloning, assembly, and maintenance of the large chromosomes in yeast. Furthermore, purification of the fragments to be assembled by electroelution greatly increased assembly efficiency.ConclusionsEntire eukaryotic chromosomes can be successfully cloned, maintained, and manipulated in yeast. These results highlight the improvement in assembly and maintenance afforded by including yeast replication origins in eukaryotic DNA with moderate G + C content (48%). They also highlight the increased efficiency of assembly that can be achieved by purifying fragments before assembly.

Marine cyanobacteria of the genus Prochlorococcus represent numerically dominant photoautotrophs residing throughout the euphotic zones in the open oceans and are major contributors to the global carbon cycle. Prochlorococcus has remained a genetically intractable bacterium due to slow growth rates and low transformation efficiencies using standard techniques. Our recent successes in cloning and genetically engineering the AT-rich, 1.1 Mb Mycoplasma mycoides genome in yeast encouraged us to explore similar methods with Prochlorococcus. Prochlorococcus MED4 has an AT-rich genome, with a GC content of 30.8%, similar to that of Saccharomyces cerevisiae (38%), and contains abundant yeast replication origin consensus sites (ACS) evenly distributed around its 1.66 Mb genome. Unlike Mycoplasma cells, which use the UGA codon for tryptophane, Prochlorococcus uses the standard genetic code. Despite this, we observed no toxic effects of several partial and 15 whole Prochlorococcus MED4 genome clones in S. cerevisiae. Sequencing of a Prochlorococcus genome purified from yeast identified 14 single base pair missense mutations, one frameshift, one single base substitution to a stop codon and one dinucleotide transversion compared to the donor genomic DNA. We thus provide evidence of transformation, replication and maintenance of this 1.66 Mb intact bacterial genome in S. cerevisiae.

We have recently developed the HB tag as a useful tool for tandem-affinity purification under native as well as fully denaturing conditions. The HB tag and its derivatives consist of a hexahistidine tag and a bacterially-derived in vivo biotinylation signal peptide, which support sequential purification by Ni 2+ -chelate chromatography and binding to immobilized streptavidin. To facilitate tagging of budding yeast proteins with HB tags, we have created a series of plasmids with various selectable markers. These plasmids allow single-step PCR-based tagging and expression under control of the endogenous promoters or the inducible GAL1 promoter. HB tagging of several budding yeast ORFs demonstrated efficient biotinylation of the HB tag in vivo by endogenous yeast biotin ligases. No adverse effects of the HB tag on protein function were observed. The HB tagging plasmids presented here are related to previously reported epitope-tagging plasmids, allowing PCR-based tagging with the same locusspecific primer sets that are used for other widely used epitope-tagging strategies. The Sequences for the described plasmids were submitted to GenBank under Accession Numbers DQ407918-pFA6a-HBH-kanMX6

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Drugs of abuse and medication reconciliation testing can benefit from analysis methods capable of detecting a broader range of drug classes and analytes. Mass spectrometry analysis of a wide variety of commonly prescribed medications and over-the-counter drugs per sample also allows for application of a drug-drug-interaction (DDI) algorithm to detect adverse drug reactions (ADRs). In order to prevent adulteration of commonly collected clinical samples such as urine, dried blood spots (DBS) present a reliable alternative. A novel method is described for qualitative and quantitative multiplex analysis of 230 parent drugs, 30 illicit drugs and 43 confirmatory metabolites by HPLC–MS-MS. This method is applicable to dried blood spot (DBS) specimens collected by volumetric absorptive microsamplers (VAMS™) and confirmable in urine specimens. A patient cohort (n = 67) providing simultaneous urine specimens and dried blood spots resulted in 100% positive predictive values (PPV) of medications or illicits confirmed by detection of a parent drug and/or its metabolite during routine medication adherence analysis. An additional 5508 dried blood spot specimens screened (n = 5575) showed 5428 (97%) with an inconsistent positive compared to the provided medication list (including caffeine, cotinine or ethanol metabolites), 29 (0.5%) with no medication list and no unexpected positive results (consistent negative), and 22 (0.4%) showed all positive results matching the provided medication list (consistent positive). A drug-drug interaction (DDI) algorithm applied to all positive results, revealed 17% with serious and 56% with moderate drug-drug interaction warnings. Comprehensive dried blood spot analysis proves a reliable alternative to urine drug testing for extended medication reconciliation, with the added advantage of detecting drug-drug-interactions.