A chemical proteomics approach for the identification of accessible antigens expressed in human kidney cancer

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.     

A novel reactive ester derivative of biotin with reduced membrane permeability for in vivo biotinylation experiments

The in vivo perfusion of rodent models of disease with biotin derivatives and the subsequent comparative proteomic analysis of healthy and diseased tissues represent a promising methodology for the identification of vascular accessible biomarkers. A novel, triply charged biotinylation reagent, NHS‐β‐Ala‐(L ‐Asp)₃‐biotin, was synthesized and validated in terms of its applicability for in vivo protein biotinylation. Compared to sulfo‐NHS‐LC‐biotin, NHS‐β‐Ala‐(L ‐Asp)₃‐biotin exhibited a reduced membrane permeability and a preferential labeling of proteins localized in compartments readily accessible in vivo from the vasculature.     

Identification of vascular surface proteins by in vivo biotinylation: a method sufficiently sensitive to detect changes in rat liver 2 weeks after partial hepatectomy

We have developed a methodology to selectively isolate and identify proteins associated with the luminal surface of blood vessels using in vivo biotinylation, streptavidin-affinity chromatography, and SDS-PAGE/LC-MS/MS. This had sufficient sensitivity to identify 32 proteins with changed expression in rat livers at 2 weeks or 5 weeks after partial hepatectomy, well after the 7 day tissue remodeling period. This method could be adapted to study other angiogenic tissues including tumors.     

In vivo protein biotinylation and sample preparation for the proteomic identification of organ- and disease-specific antigens accessible from the vasculature

Targeted delivery of bioactive molecules to diseased organs or tissues by means of binding molecules specific to markers of diseases represents a promising area of pharmaceutical intervention. The availability of markers of pathology, ideally accessible from the vasculature, is crucial for such strategies. To this aim, here we present a protocol based on terminal perfusion of mice with a reactive ester derivate of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ or tissue extracts are (i) purified on streptavidin resin in the presence of strong detergents, (ii) digested on the resin and (iii) subjected to proteomic analysis. This technology is applicable to comparative proteomic investigations of differentially expressed, accessible proteins in numerous animal models having different physiological and pathological processes.     

Improved protein sequence coverage by on resin deglycosylation and cysteine modification for biomarker discovery

Membrane proteins and secreted factors (soluble proteins or extracellular matrix components) are the targets of most monoclonal antibodies, which are currently in clinical development. These proteins are frequently post‐translationally modified, e.g. by the formation of disulfide bonds or by glycosylation, which complicates their identification using proteomics technologies. Here, we describe a novel methodology for the on resin deglycosylation and cysteine modification of proteins after in vitro, in vivo or ex vivo biotinylation. Biotinylated proteins are captured on streptavidin resin and all subsequent modifications, as well as the proteolytic digestion, which yields peptides for MS analysis, are performed on resin. Using biotinylated bovine fetuin‐A as a test protein, an improvement in sequence coverage from 7.9 to 58.7% could be shown, including the identification of all three glycosylation sites. Furthermore, a complex mixture derived from the ex vivo biotinylation of vascular structures in human kidney with cancer obtained by perfusion after surgical resection revealed almost a doubling of sequence coverage for all checked proteins when analyzed by LC‐MALDI TOF/TOF.     

Identification of specific reachable molecular targets in human breast cancer using a versatile ex vivo proteomic method

Targeting of tumoral tissues is one of the most promising approaches to improve both the efficacy and safety of anticancer treatments. The identification of valid targets, including proteins specifically and abundantly expressed in cancer lesions, is of utmost importance. Despite state-of-the-art technologies, the discovery of cancer-associated target proteins still faces the limitation, in human tissues, of antigen accessibility to suitable high-affinity ligands such as human mAb bound to bioactive molecules. Terminal perfusion of tumor-bearing mice or ex vivo perfusion of human cancer-bearing organs with a reactive biotin ester solution has successfully led to the identification of novel accessible biomarkers. This methodology is however restricted to perfusable organs, and excludes most of the tissues of interest to targeted therapies, e.g. primary breast cancer and metastases. Herein, we report on the development of a new chemical proteomic method that bypasses the perfusion step and thus offers the potential to identify accessible molecular targets in virtually all types of animal and human tissues. We have validated our new procedure by identifying biomarkers selectively expressed in human breast carcinoma. Overall, this powerful technology may lay the ground not only for custom-made therapies in cancer, but also for the development of therapies that need to be selectively delivered in a specific tissue.     

Codon optimization of the BirA enzyme gene leads to higher expression and an improved efficiency of biotinylation of target proteins in mammalian cells

Biotinylation of proteins is an attractive alternative to 'epitope-tagging', due to the strong biotin-(strept)avidin interaction and to the wide commercial availability of reagents for detection and purification of biotinylated macromolecules. Enzymatic biotinylation of target proteins in vivo using short biotin acceptor domains was described previously. Their use in mammalian cell requires expression of the bacterial biotinylation enzyme BirA. Here we describe the construction of a humanized version of BirA, with most of the rare codons replaced by codons that are more frequently used in human cells. The humanized BirA is expressed better in mammalian cells, resulting in improved efficiency of biotinylation in vivo. We anticipate that the humanized BirA gene will find use in many applications that involve in vivo biotinylation.     

A novel multi-affinity tag system to produce high levels of soluble and biotinylated proteins in Escherichia coli

We describe here a novel multi-affinity tag vector that can be used to produce high levels of soluble, in vivo biotinylated proteins in Escherichia coli. This system combines the solubility-enhancing ability of maltose-binding protein (MBP), the versatility of the hexahistidine tag (His(6)), and the site-specific in vivo biotinylation of a 15-amino acid tag (AviTag). We used this multi-tag system in an attempt to improve expression levels of two prokaryotic proteins-elongation factor Tu (TufB) and DNA gyrase subunit A (GyrA)-as well as two eukaryotic nuclear receptors-glucocorticoid receptor (GR) and small heterodimer partner (SHP). The multi-tag system not only vastly improved the expression of the two prokaryotic proteins tested, but also yielded complete, site-specific, in vivo biotinylation of these proteins. The results obtained from the TufB expression and purification are presented and discussed in detail. The nuclear receptors, though soluble as fusion partners, failed to remain soluble once the MBP tag was cleaved. Despite this limitation of the system, the multi-affinity tag approach is a useful system that can improve expression of some otherwise insoluble or poorly expressing proteins, to obtain homogeneous, purified, fully biotinylated protein for downstream applications.     

Molecular methods to study protein trafficking between organs

Interorgan communication networks are key regulators of organismal homeostasis, and their dysregulation is associated with a variety of pathologies. While mass spectrometry proteomics identifies circulating proteins and can correlate their abundance with disease phenotypes, the tissues of origin and destinations of these secreted proteins remain largely unknown. In vitro approaches to study protein secretion are valuable, however, they may not mimic the complexity of in vivo environments. More recently, the development of engineered promiscuous BirA* biotin ligase derivatives has enabled tissue-specific tagging of cellular secreted proteomes in vivo. The use of biotin as a molecular tag provides information on the tissue of origin and destination, and enables the enrichment of low-abundance hormone proteins. Therefore, promiscuous protein biotinylation is a valuable tool to study protein secretion in vivo.     

Novel comprehensive approach for accessible biomarker identification and absolute quantification from precious human tissues

The identification of specific biomarkers obtained directly from human pathological lesions remains a major challenge, because the amount of tissue available is often very limited. We have developed a novel, comprehensive, and efficient method permitting the identification and absolute quantification of potentially accessible proteins in such precious samples. This protein subclass comprises cell membrane associated and extracellular proteins, which are reachable by systemically deliverable substances and hence especially suitable for diagnosis and targeted therapy applications. To isolate such proteins, we exploited the ability of chemically modified biotin to label ex vivo accessible proteins and the fact that most of these proteins are glycosylated. This approach consists of three successive steps involving first the linkage of potentially accessible proteins to biotin molecules followed by their purification. The remaining proteins are then subjected to glycopeptide isolation. Finally, the analysis of the nonglycosylated peptides and their involvement in an in silico method increased the confident identification of glycoproteins. The value of the technique was demonstrated on human breast cancer tissue samples originating from 5 individuals. Altogether, the method delivered quantitative data on more than 400 potentially accessible proteins (per sample and replicate). In comparison to biotinylation or glycoprotein analysis alone, the sequential method significantly increased the number (≥30% and ≥50% respectively) of potentially therapeutically and diagnostically valuable proteins. The sequential method led to the identification of 93 differentially modulated proteins, among which several were not reported to be associated with the breast cancer. One of these novel potential biomarkers was CD276, a cell membrane-associated glycoprotein. The immunohistochemistry analysis showed that CD276 is significantly differentially expressed in a series of breast cancer lesions. Due to the fact that our technology is applicable to any type of tissue biopsy, it bears the ability to accelerate the discovery of new relevant biomarkers in a broad spectrum of pathologies.     

Strategies for site-specific protein biotinylation using in vitro, in vivo and cell-free systems: toward functional protein arrays

This protocol details methodologies for the site-specific biotinylation of proteins using in vitro, in vivo and cell-free systems for the purpose of fabricating functional protein arrays. Biotinylation of recombinant proteins, in vitro as well as in vivo, relies on the chemoselective reaction between cysteine-biotin and a reactive thioester group at the C-terminus of a protein generated via intein-mediated cleavage. The cell-free system utilizes low concentrations of biotin-conjugated puromycin. Unlike other approaches that require tedious and costly downstream steps of protein purification, C-terminal biotinylated proteins can be captured directly onto avidin-functionalized slides from a mixture of other cellular proteins to generate the corresponding protein array. These methods were designed to maintain the integrity and activity of proteins in a microarray format, which potentially allows simultaneous functional assays of thousands of proteins. Assuming that the target proteins have been cloned into the expression vector, transformation of bacterial strain and growth of starter culture would take approximately 2 days. Expression and in vitro protein purification and biotinylation will take approximately 3 days whereas the in vivo method would take approximately 2 days. The cell-free protein biotinylation strategy requires only 6-8 h.     

Chemical proteomic and bioinformatic strategies for the identification and quantification of vascular antigens in cancer

One avenue towards the development of more selective anti-cancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. In this context, the targeted delivery of therapeutic agents to newly-formed blood vessels (“vascular targeting”) is particularly attractive, because of the dependence of tumors on new blood vessels to sustain growth and invasion, and because of the accessibility of neo-vascular structures for therapeutic agents injected intravenously. Ligand-based vascular targeting strategies crucially rely on good-quality vascular tumor markers. Here we describe a number of established technologies for the enrichment of accessible vascular proteins based on the isolation of glycoproteins, the in vivo coating of accessible cell surfaces with colloidal silica and the in vivo perfusion with reactive ester derivatives of biotin. Label-free as well as isotopic labeling based strategies for the subsequent MS-based protein quantification are outlined. Finally, bioinformatic workflows for protein quantification are depicted aiming at assisting in the evaluation of appropriate strategies for individual projects. This review gives an overview of current chemical proteomic strategies for the enrichment and quantification of the accessible vascular proteome and helps in selecting bioinformatic strategies for data analysis and validation.     

A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli.

The high affinity binding interaction of biotin to avidin or streptavidin has been used widely in biochemistry and molecular biology, often in sensitive protein detection or protein capture applications. However, in vitro chemical techniques for protein biotinylation are not always successful, with some common problems being a lack of reaction specificity, inactivation of amino acid residues critical for protein function and low levels of biotin incorporation. This report describes an improved expression system for the highly specific and quantitative in vivo biotinylation of fusion proteins. A short 'biotinylation peptide', described previously by Schatz, is linked to the N-terminus of Escherichia coli thioredoxin (TrxA) to form a new protein, called BIOTRX. The 'biotinylation peptide' serves as an in vivo substrate mimic for E. coli biotin holoenzyme synthetase (BirA), an enzyme which usually performs highly selective biotinylation of E.coli biotin carboxyl carrier protein (BCCP). A plasmid expression vector carrying the BIOTRX and birA genes arranged as a bacterial operon can be used to obtain high level production of soluble BIOTRX and BirA proteins and, under appropriate culture conditions, BIOTRX protein produced by this system is completely biotinylated. Fusions of BIOTRX to other proteins or peptides, whether these polypeptides are linked to the C-terminus or inserted into the BIOTRX active site loop, are also quantitatively biotinylated. Both types of BIOTRX fusion can be captured efficiently on avidin/streptavidin media for purification purposes or to facilitate interaction assays. We illustrate the utility of the system by measurements of antibody and soluble receptor protein binding to BIOTRX fusions immobilized on streptavidin-conjugated BIAcore chips.     

Novel system for in vivo biotinylation and its application to crab antimicrobial protein scygonadin

BirA is a biotin ligase from Escherichia coli that specifically biotinylates a lysine side-chain within a 15-amino acid acceptor peptide (also known as Avi-tag). We developed a protocol for producing recombinant BirA ligase in E. coli for in vitro biotinylation (Li and Sousa, Prot Expr Purif, 82:162-167, 2012) in which the target protein was expressed as both thioredoxin and MBP fusions, and was released by TEV protease-mediated cleavage. The liberated ligase and the fusion proteins were enzymatically active. Based on that observation, we have now developed a novel system for in vivo biotinylation by co-expressing the Avi-tagged target protein with the MBP-BirA fusion. The effectiveness of this system was demonstrated by the successful in vivo labeling of antimicrobial protein, scygonadin. This new system shows improved efficiency compared with pre-existing one and this is likely attributed to the high expression level and solubility of the co-expressed MBP-BirA.     

Nonenzymatic biotinylation of a biotin carboxyl carrier protein: unusual reactivity of the physiological target lysine

Enzyme-catalyzed addition of biotin to proteins is highly specific. In any single organism one or a small number of proteins are biotinylated and only a single lysine on each of these proteins is modified. A detailed understanding of the structural basis for the selective biotinylation process has not yet been elucidated. Recently certain mutants of the Escherichia coli biotin protein ligase have been shown to mediate "promiscuous" biotinylation of proteins. It was suggested that the reaction involved diffusion of a reactive activated biotin intermediate, biotinoyl-5'-AMP, with nonspecific proteins. In this work the reactivity of this chemically synthesized intermediate toward the natural target of enzymatic biotinylation, the biotin carboxyl carrier protein, was investigated. The results indicate that the intermediate does, indeed, react with target protein, albeit at a significantly slower rate than the enzyme-catalyzed process. Surprisingly, analysis of the products of nonenzymatic biotinylation indicates that of five lysine residues in the protein only the physiological target side chain is modified. These results indicate that either the environment of this lysine residue or its intrinsic properties render it highly reactive to nonenzymatic biotinylation mediated by biotinoyl-5'-AMP. This reactivity may be important for its selective biotinylation in vivo.     

Identification of novel accessible proteins bearing diagnostic and therapeutic potential in human pancreatic ductal adenocarcinoma

Pancreas ductal adenocarcinoma (PDAC) remains a deadly malignancy with poor early diagnostic and no effective therapy. Although several proteomic studies have performed comparative analysis between normal and malignant tissues, there is a lack of clear characterization of proteins that could be of clinical value. Systemically reachable ("potentially accessible") proteins, suitable for imaging technologies and targeted therapies represent a major group of interest. The current study explores potentially accessible proteins overexpressed in PDAC, employing innovative proteomics technologies. In the discovery phase, potentially accessible proteins from fresh human normal and PDAC tissues were ex vivo biotinylated, isolated and identified using 2D-nano-HPLC-MS/MS method. The analysis revealed 422 up-regulated proteins in the tumor, of which 83 (including protein isoforms) were evaluated as potentially accessible. Eleven selected candidates were further confirmed as up-regulated using Western blot and multiple reaction monitoring protein quantification. Of these, transforming growth factor beta-induced (TGFBI), latent transforming growth factor beta binding 2 (LTBP2), and asporin (ASPN) were further investigated by employing large scale immunohistochemistry-based validations. They were found to be significantly expressed in a large group of clinical PDAC samples compared to corresponding normal and inflammatory tissues. In conclusion, TGFBI, LTBP2, and ASPN are novel, overexpressed, and potentially accessible proteins in human PDAC. They bear the potential to be of clinical value for diagnostic and therapeutic applications and merit further studies using in vivo models.     

Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation

The extremely tight binding between biotin and avidin or streptavidin makes labeling proteins with biotin a useful tool for many applications. BirA is the Escherichia coli biotin ligase that site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (also known as Avi-tag). As a complementary approach to in vivo biotinylation of Avi-tag-bearing proteins, we developed a protocol for producing recombinant BirA ligase for in vitro biotinylation. The target protein was expressed as both thioredoxin and MBP fusions, and was released from the corresponding fusion by TEV protease. The liberated ligase was separated from its carrier using HisTrap HP column. We obtained 24.7 and 27.6 mg BirA ligase per liter of culture from thioredoxin and MBP fusion constructs, respectively. The recombinant enzyme was shown to be highly active in catalyzing in vitro biotinylation. The described protocol provides an effective means for making BirA ligase that can be used for biotinylation of different Avi-tag-bearing substrates.     

Leishmania mexicana amazonensis: development of a peptide tag useful for labeling and purifying biotinylated recombinant proteins

A number of peptide tags are available to facilitate the characterization of recombinant proteins. We have tested the bacterial oxaloacetate decarboxylase biotinylation domain for its efficacy in tagging recombinant proteins in vivo in Leishmania. To achieve efficient biotinylation, Leishmania also had to be co-transformed with the gene for bacterial biotin protein ligase (birA gene product). The recombinant chimeric protein could be detected on blots probed with avidin-horseradish peroxidase and purified on immobilized monomeric avidin resins.     

TGF-alpha-driven tumor growth is inhibited by an EGF receptor tyrosine kinase inhibitor.

The simultaneous presence of the EGFR and its ligand TGF-alpha in human tumor tissues suggests that autocrine TGF-alpha stimulation drives tumor growth. Here we show that autocrine TGF-alpha stimulation does cause increased tumor growth in vivo, an effect that was proven to be mediated via EGFR activation, and that this TGF-alpha/EGFR autocrine loop was accessible to an EGFR specific tyrosine kinase inhibitor. Clones of the EGFR expressing glioma cell line U-1242 MG were transfected with TGF-alpha cDNA using a tetracycline-inhibitory system for gene expression. TGF-alpha expression was inhibited by the presence of tetracycline, and subcutaneous tumors forming from cell lines injected into nude mice could be inhibited by feeding mice tetracycline. We confirmed that TGF-alpha mRNA and protein were present in these tumors and that, subsequently, the endogenous EGFR was activated. Tumor growth could be inhibited by an EGFR specific tyrosine kinase inhibitor of the type 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline, administered daily by intraperitoneal injection, thereby interrupting the autocrine loop.     

PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo

The common techniques to study protein-protein proximity in vivo are not well adapted to the capabilities and the expertise of a standard proteomics laboratory, typically based on the use of mass spectrometry. With the aim of closing this gap, we have developed PUB-MS (for proximity utilizing biotinylation and mass spectrometry), an approach to monitor protein-protein proximity, based on biotinylation of a protein fused to a biotin-acceptor peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The biotinylation status of the BAP can be further detected by either Western analysis or mass spectrometry. The BAP sequence was redesigned for easy monitoring of the biotinylation status by LC-MS/MS. In several experimental models, we demonstrate that the biotinylation in vivo is specifically enhanced when the BAP- and BirA-fused proteins are in proximity to each other. The advantage of mass spectrometry is demonstrated by using BAPs with different sequences in a single experiment (allowing multiplex analysis) and by the use of stable isotopes. Finally, we show that our methodology can be also used to study a specific subfraction of a protein of interest that was in proximity with another protein at a predefined time before the analysis.