Differential dimethyl labeling of N-termini of peptides after guanidination for proteome analysis

We describe an enabling technique for proteome analysis based on isotope-differential dimethyl labeling of N-termini of tryptic peptides followed by microbore liquid chromatography (LC) matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS). In this method, lysine side chains are blocked by guanidination to prevent the incorporation of multiple labels, followed by N-terminal labeling via reductive amination using d(0),(12)C-formaldehyde or d(2),(13)C-formaldehyde. Relative quantification of peptide mixtures is achieved by examining the MALDI mass spectra of the peptide pairs labeled with different isotope tags. A nominal mass difference of 6 Da between the peptide pair allows negligible interference between the two isotopic clusters for quantification of peptides of up to 3000 Da. Since only the N-termini of tryptic peptides are differentially labeled and the a(1) ions are also enhanced in the MALDI MS/MS spectra, interpretation of the fragment ion spectra to obtain sequence information is greatly simplified. It is demonstrated that this technique of N-terminal dimethylation (2ME) after lysine guanidination (GA) or 2MEGA offers several desirable features, including simple experimental procedure, stable products, using inexpensive and commercially available reagents, and negligible isotope effect on reversed-phase separation. LC-MALDI MS combined with this 2MEGA labeling technique was successfully used to identify proteins that included polymorphic variants and low abundance proteins in bovine milk. In addition, by analyzing a mixture of two equal amounts of milk whey fraction as a control, it is shown that the measured average ratio for 56 peptide pairs from 14 different proteins is 1.02, which is very close to the theoretical ratio of 1.00. The calculated percentage error is 2.0% and relative standard deviation is 4.6%.     

Quantitation using enhanced signal tags: a technique for comparative proteomics

Differential amidination of N-termini and lysine residues provides the basis for a novel approach to protein quantitation using MALDI mass spectrometry. Because the amidination of lysines increases their basicity and therefore MALDI ionization yields, the method is called quantitation using enhanced signal tags (QUEST). Amidine labels differ by methylene groups, leading to 14 Da mass differentials. The utility of QUEST is demonstrated while analyzing the digests of two model proteins using MALDI-TOF mass spectrometry.     

A calcium-dependent antibody for identification and purification of recombinant proteins

We report a straightforward methodology for purification of recombinant proteins by incorporating a short hydrophilic peptide marker segment at their N-termini. A calcium-dependent antibody that reacts primarily with the first three amino acids of this peptide segment was used to affinity purify the fusion proteins in a single chromatographic step. The marker peptide could subsequently be removed by proteolysis with the enzyme enterokinase.     

Gapped spectral dictionaries and their applications for database searches of tandem mass spectra

Generating all plausible de novo interpretations of a peptide tandem mass (MS/MS) spectrum (Spectral Dictionary) and quickly matching them against the database represent a recently emerged alternative approach to peptide identification. However, the sizes of the Spectral Dictionaries quickly grow with the peptide length making their generation impractical for long peptides. We introduce Gapped Spectral Dictionaries (all plausible de novo interpretations with gaps) that can be easily generated for any peptide length thus addressing the limitation of the Spectral Dictionary approach. We show that Gapped Spectral Dictionaries are small thus opening a possibility of using them to speed-up MS/MS searches. Our MS-Gapped-Dictionary algorithm (based on Gapped Spectral Dictionaries) enables proteogenomics applications (such as searches in the six-frame translation of the human genome) that are prohibitively time consuming with existing approaches. MS-Gapped-Dictionary generates gapped peptides that occupy a niche between accurate but short peptide sequence tags and long but inaccurate full length peptide reconstructions. We show that, contrary to conventional wisdom, some high-quality spectra do not have good peptide sequence tags and introduce gapped tags that have advantages over the conventional peptide sequence tags in MS/MS database searches.     

New trends in aggregating tags for therapeutic protein purification

The rapid growth of the therapeutic protein market calls for more efficient purification methods. Various aggregating tags have recently emerged as simple, fast, cost-effective and column-free technologies for protein (and peptide) purification. In general, these column-free protein purification technologies involve the use of aggregating tags that induce the target protein into insoluble aggregates. These aggregates can be easily separated from soluble impurities and the target protein or peptide is then liberated by a cleavage process. This review summarizes the current state-of-the-art in using aggregating tags for protein purification. The methods are here categorized as follows: (1) tags that allow soluble expression of target protein in vivo and induce aggregation in vitro; (2) tags that induce soluble expression and self-assembling of target protein on insoluble biological polyester beads in vivo; (3) tags that induce formation of inactive aggregates in vivo; (4) tags that induce formation of active aggregates in vivo.     

Comparison of affinity tags for protein purification

Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.     

Development of iodoacetic acid-based cysteine mass tags: detection enhancement for cysteine-containing peptide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We developed and characterized 6 new cysteine mass tags for high-sensitivity peptide analysis. The structural features are: (1) iodoacetyl group for thiol tagging, (2) hydrophilic character for reducing sample loss, (3) tertiary amino, quaternary ammonium, or guanidino group for high proton affinity, and (4) no amide bonding for minimizing fragmentation of tag moiety in collision-induced dissociation. By using these tags, 2- to 200-fold MS sensitivity was achieved, compared to control peptide with carbamydomethylation.     

STEM: a software tool for large-scale proteomic data analyses

We describe the software, STEM (STrategic Extractor for Mascot's results), which efficiently processes large-scale mass spectrometry-based proteomics data. V (View)-mode evaluates the Mascot peptide identification dataset, removes unreliable candidates and redundant assignments, and integrates the results with key information in the experiment. C (Comparison)-mode compares peptide coverage among multiple datasets and displays proteins commonly/specifically found therein, and processes data for quantitative studies that utilize conventional isotope tags or tags having a smaller mass difference. STEM significantly improves throughput of proteomics study.     

MINLP models for the synthesis of optimal peptide tags and downstream protein processing

The development of systematic methods for the synthesis of downstream protein processing operations has seen growing interest in recent years, as purification is often the most complex and costly stage in biochemical production plants. The objective of the work presented here is to develop mathematical models based on mixed integer optimization techniques, which integrate the selection of optimal peptide purification tags into an established framework for the synthesis of protein purification processes. Peptide tags are comparatively short sequences of amino acids fused onto the protein product, capable of reducing the required purification steps. The methodology is illustrated through its application on two example protein mixtures involving up to 13 contaminants and a set of 11 candidate chromatographic steps. The results are indicative of the benefits resulting by the appropriate use of peptide tags in purification processes and provide a guideline for both optimal tag design and downstream process synthesis.     

Peptides from the SARS-associated coronavirus as tags for protein expression and purification

Protein tagging with a peptide is a commonly used technique to facilitate protein detection and to carry out protein purification. Flexibility with respect to the peptide tag is essential since no single tag suites all purposes. This report describes the usage of two short peptides from the SARS-associated coronavirus nucleocapsid (SARS-N) protein as protein tags. Plasmids for the generation of tagged proteins were generated by ligating synthetic oligonucleotides for the peptide-coding regions downstream of the protein coding sequence. The data show recognition of prokaryotically expressed HIV-1 Gag/p24 fusion protein by Western blot and efficient affinity purification using monoclonal antibodies against the tags. The SARS peptide antibody system described presents an alternative tagging opportunity in the growing field of protein science.     

The p1 protein of the yeast transposon Ty1 can be used for the construction of bi-functional virus-like particles

Virus-like particles (VLPs) containing heterologous proteins are often used as vaccines. Two approaches for the construction of bi-functional VLPs using hybrid protein pl-380 of the TY1 transposon of Saccharomyces yeast are described. We have shown that both C- and N-termini of p1-380 can be used for the expression of heterologous peptides. Peptides from A. Fumigatus Asp f 2, expressed at the C- and/or N-termini of p1-380, did not interfere with VLP self-assembling, were accessible for antibodies and hence were exposed at the VLP surface. Another way to obtain bivalent VLPs is the formation of mixed particles, which co-express two hybrid pl proteins with different heterologous protein fragments at the C-terminus. To do it the yeast cells were transfected with a mixture of two recombinant DNA coding Asp f 2 peptide and green fluorescent protein (Gfp). We have shown that both Asp f 2 peptide and Gfp are expressed within the same particle. To evaluate biological activity of bi-functional VLP a construction containing peptides representing dominant T- and B-cell epitopes of Asp f 2 was produced. Bi-functional particles were more potent in stimulating memory immune responses. These results demonstrate new possibilities of pl-380 based expression system to produce multifunctional VLPs.     

Hydrophobic peptide tags as tools in bioseparation

Hydrophobic interactions are highly selective, and differences in surface hydrophobicities between proteins can be used as an efficient handle to facilitate protein isolation. Aromatic amino acid residues are of particular importance for molecular recognition because they have a key role in several biological functions. The hydrophobicity of a protein can easily be altered with minor genetic modifications, such as site-directed mutagenesis or fusions of hydrophobic peptide tags. An important advantage of hydrophobic peptide tags over traditional affinity tags is the possibility of exploring simple and inexpensive bioseparation materials. Recent results demonstrate the potential of hydrophobic interaction chromatography and aqueous two-phase systems as tools to study relative hydrophobicities of recombinant proteins with only minor alterations. This review focuses on hydrophobic peptide tags as fusion partners, which can be used as important tools in bioseparation.     

Binary polypeptide system for permanent and oriented protein immobilization

Background  

Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling.     

Optimisation of a multivalent Strep tag for protein detection

The Strep tag is a peptide sequence that is able to mimic biotin's ability to bind to streptavidin. Sequences of Strep tags from 0 to 5 have been appended to the N-terminus of a model protein, the Stefin A Quadruple Mutant (SQM) peptide aptamer scaffold, and the recombinant fusion proteins expressed. The affinities of the proteins for streptavidin have been assessed as a function of the number of tags inserted using a variety of labelled and label-free bioanalytical and surface based methods (Western blots, microarray assays and surface plasmon resonance spectroscopy). The binding affinity increases with the number of tags across all assays, reaching nanomolar levels with 5 inserts, an observation assigned to a progressive increase in the probability of a binding interaction occurring. In addition a novel interfacial FRET based assay has been developed for generic Strep tag interactions, which utilises a conventional microarray scanner and bypasses the requirement for expensive lifetime imaging equipment. By labelling both the tagged StrepX-SQM(2) and streptavidin targets, the conjugate is primed for label-free FRET based displacement assays.     

New protein purification system using gold-magnetic beads and a novel peptide tag, "the methionine tag"

Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.     

Do-it-yourself histidine-tagged bovine enterokinase: A handy member of the protein engineer's toolbox

Enterokinase, a two-chain duodenal serine protease, activates trypsinogen by removing its N-terminal propeptide. Due to a clean cut after the non-primed site recognition sequence, the enterokinase light chain is frequently employed in biotechnology to separate N-terminal affinity tags from target proteins with authentic N-termini. In order to obtain large quantities of this protease, we adapted an in vitro folding protocol for a pentahistidine-tagged triple mutant of the bovine enterokinase light chain. The purified, highly active enzyme successfully processed recombinant target proteins, while the pentahistidine-tag facilitated post-cleavage removal. Hence, we conclude that producing enterokinase in one's own laboratory is an efficient alternative to the commercial enzyme.     

Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis.

The genes coding for the photosynthetic reaction center cytochrome c subunit (pufC) and the soluble cytochrome c2 (cycA) from the purple non-sulfur bacterium Rhodopseudomonas viridis were expressed in Escherichia coli. Biosynthesis of the reaction center cytochrome without a signal peptide resulted in the formation of inclusion bodies in the cytoplasm amounting to 14% of the total cellular protein. A series of plasmids coding for the cytochrome subunit with varying N-terminal signal peptides was constructed in attempts to achieve translocation across the E. coli cytoplasmic membrane and heme attachment. However, the two major recombinant proteins with N-termini corresponding to the signal peptide and the cytochrome were synthesized in E. coli as non-specific aggregates without heme incorporation. An increased ratio of precursor as compared to 'processed' apo-cytochrome was obtained when expression was carried out in a proteinase-deficient strain. Cytochrome c2 from R. viridis was synthesized in E. coli as a precursor associated with the cytoplasmic membrane. An expression plasmid was designed encoding the N-terminal part of the 33 kDa precursor protein of the oxygen-evolving complex of Photosystem II from spinach followed by cytochrome c2. Two recombinant proteins without heme were found to aggregate as inclusion bodies with N-termini corresponding to the signal peptide and the mature 33 kDa protein.     

Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases

We have developed a specific and efficient method for complete removal of polyhistidine purification tags (HisTags) from the N-termini of target proteins. The method is based on the use of the aminopeptidase dipeptidyl peptidase I (DPPI), either alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP). In both cases, the HisTag is cleaved off by DPPI, which catalyzes a stepwise excision of a wide range of dipeptides from the N-terminus of a peptide chain. Some sequences, however, are resistant to DPPI cleavage and a number of mature proteins have nonsubstrate N-termini which protects them against digestion. For such proteins, HisTags composed of an even number of residues can be cleaved off by treatment with DPPI alone. When the target protein is unprotected against DPPI, a blocking group is generated enzymatically from a glutamine residue inserted between the HisTag and the target protein. A protein with a HisTag-Gln extension is incubated with both DPPI and GCT. As above, the polyhistidine sequence is cleaved off by DPPI, but when the glutamine residue appears in the N-terminus, it is immediately converted into a pyroglutamyl residue by an excess of GCT and further DPPI digestion is prevented. The desired sequence is finally obtained by excision of the pyroglutamyl residue with PGAP. All the enzymes employed can bind to immobilized metal affinity chromatography (IMAC) matrices, and in this paper we demonstrate a simple and highly effective process combining IMAC purification of His-tagged proteins, our aminopeptidase-based method for specific excision of HisTags and use of subtractive IMAC for removing processing enzymes. Typical recoveries were 75-90% for the enzymatic processing and subtractive IMAC. The integrated process holds promises for use in large-scale production of pharmaceutical proteins because of a simple overall design, use of robust and inexpensive matrices, and use of enzymes of either recombinant or plant origin.     

Protein Purification with C-Terminal Fusion of Maltose Binding Protein

For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity tags are needed which can be fused to the C-terminus. In this publication we describe that maltose binding protein (MBP) fused to the C-terminus of the plant photoreceptor phytochrome B allows purification of the fusion protein via amylose affinity chromatography. After overexpression in yeast a 125-fold enrichment could be achieved. The spectral properties of phytochrome B were not impaired by the fusion and purification. These results demonstrate that not only the widely used N-terminal fusions of MBP but also C-terminal fusions can be employed for protein purification.     

SPIDER: software for protein identification from sequence tags with de novo sequencing error

For the identification of novel proteins using MS/MS, de novo sequencing software computes one or several possible amino acid sequences (called sequence tags) for each MS/MS spectrum. Those tags are then used to match, accounting amino acid mutations, the sequences in a protein database. If the de novo sequencing gives correct tags, the homologs of the proteins can be identified by this approach and software such as MS-BLAST is available for the matching. However, de novo sequencing very often gives only partially correct tags. The most common error is that a segment of amino acids is replaced by another segment with approximately the same masses. We developed a new efficient algorithm to match sequence tags with errors to database sequences for the purpose of protein and peptide identification. A software package, SPIDER, was developed and made available on Internet for free public use. This paper describes the algorithms and features of the SPIDER software.