Oligomeric ethylene glycols as sorting tags for parallel and combinatorial mixture synthesis

Sorting tags for chromatography allow mixtures of substrates to be carried through parallel chemical processes and then separated. The first sorting tags were fluorocarbons. An enabling characteristic of fluorous sorting tags is the predictable incremental increase in retention time with increasing fluorocarbon chain length. Here we describe a general approach to sorting tags and report our discovery of a second class of chromatographic tags. The new tags are oligomeric ethylene glycol (OEG) derivatives.     

Recyclable benzyl-type fluorous tags: Preparation and application in oligosaccharide synthesis

We herein described the design, synthesis and application of two recyclable benzyl-type fluorous tags with double fluorous chains. The benzyl-type fluorous tags were prepared in 3 steps from a commercially available fluorous alcohol. The glycosylation of the benzyl-type tags with imidate donors proceeded smoothly to provide the corresponding fluorous-tagged carbohydrates in good to excellent yields, which were readily purified by fluorous solid-phase extraction(FSPE). Efficient removal of the tags from tagtethered carbohydrates were conducted under the common catalytic hydrogenation condition and the initial benzyl-type fluorous tags could be regenerated [5_TD$IF]via a 2-step simple procedure in 69%–93% yields.The utility of the new benzyl-fluorous tag was demonstrated [7_TD$IF]via the FSPE-assisted synthesis of oligosaccharides Gb3.     

Aliphatic dipeptide tags for multi-2-plex protein quantification

Mass-balanced (1)H/(2)H-isotope dipeptide tag (MBIT) is diversified as aliphatic tags for multiplexed protein quantification. Aliphatic MBITs are based on the N-acetyl-Xxx-Ala dipeptide, where Xxx is an artificial amino acid with a linear alkyl side chain from C(2)H(5) to C(8)H(17) (C(2)-C(8) tags). (1)H/(2)H isotopes are encoded in the methyl groups of N-acetyl and Ala to yield a pair of isobaric tags with 2-plex quantitation signals separated by 3 Da. C(2)-C(5) tags are prepared by solid-phase synthesis, while C(6)-C(8) tags are synthesized by olefin metathesis in solution. These aliphatic tags are made reactive toward the primary amines of peptides, and the relative abundances of quantitation signals are characterized using both matrix-assisted laser desorption ionization and electrospray ionization tandem mass spectrometry. MBIT-linked peptides co-migrate in reverse-phase liquid chromatography (LC), and their tandem mass spectra exhibit 2-plex quantitation signals as well as sequence ions in similar abundances. As the length of alkyl side chain increases, C(2)-C(8) tags show a stepwise increase in both the LC retention time and the relative abundance of quantitation signals. In addition, the quantitation linearity is well-maintained in a 15-250 fmol range. The multiplexing capability of aliphatic MBITs is demonstrated by applying three different tags (C(6)-C(8) tags) to the quantification of yeast heat shock proteins expressed under four different physiological conditions.     

Novel chemical tagging method for combinatorial synthesis utilizing suzuki chemistry and fourier transform ion cyclotron resonance mass spectrometry

We describe a novel chemical tagging strategy for combinatorial solid-phase chemistry. The tags used are para-substituted alkyl phenols, with the first tags attached directly to the chloromethyl polystyrene and subsequent tags attached via Suzuki couplings using either aryl diboronic acids or aryl iodides. The identities of the tags attached to a single bead are discovered by the high-resolution, accurate mass technique of Fourier transform ion cyclotron resonance mass spectrometry. The method is exemplified for the coded assembly of a tripeptide.     

Rapid evaluation of nickel binding properties of His-tagged lactate dehydrogenases using surface plasmon resonance

The use of surface plasmon resonance (SPR), for the comparison of metal binding properties of polyhistidine tags, was evaluated. Six different tags containing various number of histidines, either none (tags n and t), three (tags H3A3 and HA2HA2H) or six (tags H6 and His6), were genetically fused to the N-terminal of lactate dehydrogenase (LDH). The binding ability of these constructs to nickel ions, immobilised with nitrilotriacetic acid (NTA), was tested both by conventional immobilised metal ion affinity chromatography (IMAC) and SPR. The relative binding strengths of the tags to nickel were identical using both methods (n approximately t < HA2HA2H < H3A3 < His6 < H6), confirming the value of the SPR technique for investigating metal-protein interactions. Protein modelling has also proved to be useful in supporting the experimental results.     

Rewritable remote encoding and decoding of miniature multi-bit magnetic tags for high-throughput biological analysis

We have investigated a new magnetic labelling technology for high-throughput biomolecular identification and DNA sequencing. Planar multi-bit magnetic tags comprising a magnetic barcode formed by an ensemble of micron-sized thin film ferromagnetic Co bars and a 15 x 15 micron Au square for immobilization of probe molecules have been designed and fabricated. We show that by using a globally applied magnetic field and magneto-optical Kerr microscopy the magnetic elements in the multi-bit magnetic tags can be addressed individually and encoded/decoded remotely. The power of the approach is the read/write technique, which allows modest globally applied magnetic fields to write almost unlimited numbers of codes to populations of tags rather than individuals. The magnetic nature of the technology also lends itself naturally to fast, remote decoding and the ability to rewrite tags if needed. We demonstrate the critical steps needed to show the feasibility of this technology, including fabrication, remote writing and reading, and successful functionalization of the tags as verified by fluorescence detection. This approach is ideal for encoding information on tags in microfluidic flow or suspension, in order to label oligonucleotides during split-and-mix synthesis, and for combinatorial library-based high-throughput multiplexed bioassays.     

Bioinspired Nitroalkylation for Selective Protein Modification and Peptide Stapling

Nitroalkanes react specifically with aldehydes, providing rapid, stable, and chemoselective protein bioconjugation. These nitroalkylated proteins mimic key post‐translational modifications (PTMs) of proteins and can be used to understand the role of these PTMs in cellular processes. Demonstrated here is the substrate scope of this bioconjugation by attaching a variety of tags, such as NMR tags, fluorescent tags, affinity tags, and alkyne tags, to proteins. The structure and enzymatic activity of modified proteins remain conserved after labeling. Notably, the nitroalkane group leads to easy characterization of proteins by mass spectrometry because of its distinct fingerprint pattern. Importantly, the nitro‐alkylated peptides provide a new handle for site‐selective fluorination of peptides, thus installing a specific probe to study peptide–protein interactions by ¹⁹F NMR spectroscopy. Furthermore, nitroalkane reagents can be used for the late‐stage diversification of peptides and for the synthesis of peptide staples.     

1,3,5-triazine-based mass spectral tagging of one-bead one-compound libraries

A triazine-based mass encoding strategy that accommodates cleavable linker, isotopic labeling, and diversity receptor moieties is reported. The resulting triazine-based tags, which are coupled to bifunctionalized TentaGel resin in a one-pot transformation, enable the construction of a 1-oxa-2,8-diazaspiro[4.5]dec-2-ene-7-carboxamide library and facilitate decoding by equalizing the ionization potential of the liberated tags in single bead MALDI-TOF experiments as well as balancing the reactivity of the starting tags in the resin coupling step. [reaction: see text].     

"Mass defect" tags for biomolecular mass spectrometry

We present a new class of "mass defect" tags with utility in biomolecular mass spectrometry. These tags, incorporating element(s) with atomic numbers between 17 (Cl) and 77 (Ir), have a substantially different nuclear binding energy (mass defect) from the elements common to biomolecules. This mass defect yields a readily resolvable mass difference between tagged and untagged species in high-resolution mass spectrometers. We present the use of a subset of these tags in a new protein sequencing application. This sequencing technique has advantages over existing mass spectral protein identification methodologies: intact proteins are quickly sequenced and unambiguously identified using only an inexpensive, robust mass spectrometer. We discuss the potential broader utility of these tags for the sequencing of other biomolecules, differential display applications and combinatorial methods.     

Rigid,Highly Reactive and Stable DOTA-like Tags Containing a Thiol-Specific Phenylsulfonyl Pyridine Moiety for Protein Modification and NMR Analysis**

Site specific installation of a paramagnetic ion with magnetic anisotropy in a biomolecule generates valuable structural restraints, such as pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs). These paramagnetic effects can be used to characterize the structures, interactions and dynamics of biological macromolecules and their complexes. Two single-armed DOTA-like tags, BrPSPy-DO3M(S)A-Ln and BrPSPy-6M-DO3M(S)A-Ln, each containing a thiol-specific reacting group, that is, a phenylsulfonyl pyridine moiety, are demonstrated as rigid, reactive and stable paramagnetic tags for protein modification by formation of a reducing resistant thioether bond between the protein and the tag. The two tags present high reactivity with the solvent exposed thiol group in aqueous solution at room temperature. The introduction of Br at the meta-position in pyridine enhances the reactivity of 4-phenylsulfonyl pyridine towards the solvent exposed thiol group in a protein, whereas the ortho-methyl group in pyridine increases the rigidity of the tag in the protein conjugates. The high performance of these two tags has been demonstrated in different cysteine mutants of ubiquitin and GB1. The high reactivity and rigidity of these two tags can be added in the toolbox of paramagnetic tags suitable for the high-resolution NMR measurements of biological macromolecules and their complexes.     

Comparative analysis of cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics

The advances in bioorthogonal ligation methods have provided new opportunities for proteomic analysis of newly synthesized proteins, posttranslational modifications, and specific enzyme families using azide/alkyne-functionalized chemical reporters and activity-based probes. Efficient enrichment and elution of azide/alkyne-labeled proteins with selectively cleavable affinity tags are essential for protein identification and quantification applications. Here, we report the synthesis and comparative analysis of Na?S?O?-cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. We demonstrated that ortho-hydroxyl substituent is required for efficient azobenzene-bond cleavage and show that these cleavable affinity tags can be used to identify newly synthesized proteins in bacteria targeted by amino acid chemical reporters as well as their sites of modification on endogenously expressed proteins. The azobenzene-based affinity tags are compatible with in-gel, in-solution, and on-bead enrichment strategies and should afford useful tools for diverse bioorthogonal proteomic applications.     

Combination of Mass Signal Amplification and Isotope‐Labeled Alkanethiols for the Multiplexed Detection of miRNAs

We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope‐labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am‐tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am‐tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am‐tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO₄ concentrations.     

Raman reporter-coated gold nanorods and their applications in multimodal optical imaging of cancer cells

We report the preparation of a kind of surface-enhanced Raman scattering (SERS) tags and explore their applications in multifunctional optical imaging of cancer cells. The proposed nanoparticles (SERS tags) are prepared by connecting dye molecules directly onto the surfaces of gold nanorods through Au–S or Au–N interactions. The dye molecules are used as Raman reporters, while gold nanorods are used as enhanced materials due to their localized surface plasmon resonance effect. Multilayered polymers are further coated onto the surfaces of the nanoparticles to reach better stability and biocompatibility. Gold nanorods with different aspect ratios and different dye molecules conjugated are compared in order to achieve the diversity of SERS tags and find out the optimized condition of SERS tags with the highest signal intensity. Our experiments show that the resulting nanoparticles, which are uptaken by cancer cells, can provide not only dark field cells images but also multiplexing SERS images.     

Evaluation of fully-functionalized diazirine tags for chemical proteomic applications

The use of photo-affinity reagents for the mapping of noncovalent small molecule–protein interactions has become widespread. Recently, several ‘fully-functionalized’ (FF) chemical tags have been developed wherein a photoactivatable capture group, an enrichment handle, and a functional group for synthetic conjugation to a molecule of interest are integrated into a single modular tag. Diazirine-based FF tags in particular are increasingly employed in chemical proteomic investigations; however, despite routine usage, their relative utility has not been established. Here, we systematically evaluate several diazirine-containing FF tags, including a terminal diazirine analog developed herein, for chemical proteomic investigations. Specifically, we compared the general reactivity of five diazirine tags and assessed their impact on the profiles of various small molecules, including fragments and known inhibitors revealing that such tags can have profound effects on the proteomic profiles of chemical probes. Our findings should be informative for chemical probe design, photo-affinity reagent development, and chemical proteomic investigations.

The chemical proteomic properties of five diazirine-based, fully-functionalized photoaffinity tags, including a newly developed, minimal tag, were compared. This study provides guidance for the development of new photoaffinity probes.     

Generating protein sequence tags by combining cone and conventional collision induced dissociation in a quadrupole time-of-flight mass spectrometer

The goal of proteomics research is to be able to identify and quantify the vast numbers of proteins within an organism or tissue. "Top-down" methods address this goal without the need for proteolytic digestion prior to mass analysis. We report here an approach for top-down protein identification that has been implemented on a commercially available, unmodified Qq-TOF mass spectrometer. Intact protein molecular ions first undergo cone fragmentation in the electrospray inlet. Conventional MS/MS is then performed on a mass selected cone fragment using CID in the Qq interface of the Qq-TOF mass spectrometer to generate a sequence tag through a pseudo-MS3 experiment. Seven proteins varying in molecular weight between 11 and 66 kDa were chosen to demonstrate applicability of method. After the molecular weight of the intact protein was determined, the cone voltage was varied to induce fragmentation. Cone fragment ions were then further dissociated using conventional CID, and the resulting MS/MS spectra were processed and analyzed for sequence tags. Sequence tags were easily identified from a MS/MS spectrum of a cone induced fragment ion both manually and through a de novo sequencing program included in the software associated with the mass spectrometer. Sequence tags were subjected to database searching using the PeptideSearch program of EMBL, and all protein sequence tags gave unambiguous search results. In all cases, sequence tags were found to originate from the n- and/or c-termini of the proteins.     

Metal ion affinity purification of proteins by genetically incorporating metal-chelating amino acids

Affinity tags are efficient tools for protein purification. They allow simple one-step purification of proteins to high purity. However, in some cases the tags cause structural and functional changes in a protein, and need to be removed. Therefore, affinity tags that are readily introduced into proteins with minimal perturbation and have specific affinity for purification are desired. Herein, two metal-chelating amino acids derived from 2,2′-bipyridine and 8-hydroxyquinoline were genetically incorporated into glutathione S-transferase (GST) and the mutant proteins were purified by using the metal ion affinity of the unnatural amino acids. The purification of the GST mutants containing 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) showed that the proteins could be efficiently enriched in Ni–NTA by the metal ion affinity of the unnatural amino acid and purified to excellent purity. This method should be very useful for general protein affinity purification, especially for proteins whose structure or function is affected by affinity tags fused to N- or C-terminals.     

Screening of peptide affinity tags using immobilised metal affinity chromatography in 96-well plate format

A method for high throughput screening of Green Fluorescent Proteins carrying metal binding tags in bacteria was developed. A random four amino acids tag-peptide library was successfully generated in E. coli. A 96-microtiter plate assembled with metal-iminodiacetic acid small cryogel columns was used for library screening. For the first time we were able to simultaneously screen a metal binding peptide tags library obtained from E. coli against different metal ions. From screening 25 different tags, three clones were able to bind to all metal ions studied (Ni2+, Zn2+, Co2+ and Cd2+). It was clearly demonstrated that the new construct could facilitate the screening of large peptide libraries.     

Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.     

Adsorptive detagging of poly-histidine tagged protein using hexa-histidine tagged exopeptidase

The ubiquitous use of poly-histidine fusion tags has made the purification of the recombinant target proteins much simpler, although the presence of residual fusion tags can generate immunogenic products or products with changed biological activities. This work presents a generic method of removing poly-histidine fusion tags from recombinant proteins through the use of a hexa-histidine tagged exopeptidase (DAPase) when both tagged species are adsorbed to the immobilized metal affinity chromatography (IMAC) adsorbent. Adsorptive detagging was performed in the presence of 50mM imidazole in order to allow the cleavage reaction by the hexa-histidine tagged DAPase to occur. The progress of batch and adsorptive detagging by DAPase of maltose binding protein (MBP) tagged with two variants of hexa-histidine fusion tag was successfully monitored using cationic exchange chromatography. A single-step, column-based detagging strategy was then optimized to maximize the recovery of native MBP. The kinetics of batch and on-column digestion for both HT6 and HT15 fusion tags were investigated. The process involved the sequential removal of dipeptides during the digestion of full-length fusion protein down to its fully detagged native form. During the course of tag digestion, 4 and 7 different intermediates were detected for HT6 and HT15 tagged MBP respectively. The characteristics of on-column cleavage of poly-histidine fusion tags by DAPase as a function of incubation temperature and amount of protease activity used were examined. It was found that the influence of fusion tag design on the batch and column-based detagging yield and efficiency was substantial. In addition, the structural difference of fusion tags affects the binding strength of the fusion protein, which can influence the resulting product purity. Despite being a longer tag, HT15 fusion tag was the preferred sequence for shortening the time needed for on-column detagging. These results can be applied to the wider use of the proposed platform protocol for the on-column cleavage of poly-histidine tagged proteins using exopeptidases.     

Phosphonium ion tagged chiral phosphoric acids and their application in Friedel-Crafts reactions of indoles

The attachment of phosphonium ion phase tags to chiral binapthyl-based phosphoric acid catalysts, and the use of these materials in a range of organocatalytic asymmetric Friedel-Crafts reactions of indoles has been studied. Placement of the tags at the 3 and 3′ positions of the phosphoric acid, so that they could serve as steric blocking groups, failed to produce an active catalyst. However, moving the phosphonium ion groups to the 6 and 6′ positions produced an efficient and enantioselective catalyst. Aided by the presence of the phase tags, the chiral catalyst was easily removed at the end of the reactions, and could be reused several times, albeit with somewhat decreased efficiency and enantioselectivity.