Josip Blonder

Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS

The core prerequisites for an efficient proteome-scale analysis of mammalian membrane proteins are effective isolation, solubilization, digestion and multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). This protocol is for analysis of the mammalian membrane proteome that relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (vol/vol) methanol. Tryptic digestion is followed by strong cation exchange (SCX) chromatography and reversed phase (RP) chromatography coupled online with MS/MS for protein identification. The use of a methanol-based buffer eliminates the need for reagents that interfere with chromatographic resolution and ionization of the peptides (e.g., detergents, chaotropes, inorganic salts). Sample losses are minimized because solubilization and digestion are carried out in a single tube avoiding any sample transfer or buffer exchange between these steps. This protocol is compatible with stable isotope labeling at the protein and peptide level, enabling identification and quantitation of integral membrane proteins. The entire procedure—beginning with isolated membrane fraction and finishing with MS data acquisition—takes 4–5 d.

Global Analysis of the Cortical Neuron Proteome

In this study, a multidimensional fractionation approach was combined with MS/MS to increase the capability of characterizing complex protein profiles of mammalian neuronal cells. Proteins extracted from primary cultures of cortical neurons were digested with trypsin followed by fractionation using strong cation exchange chromatography. Each of these fractions was analyzed by microcapillary reversed-phase LC-MS/MS. The analysis of the MS/MS data resulted in the identification of over 15,000 unique peptides from which 3,590 unique proteins were identified based on protein-specific peptide tags that are unique to a single protein in the searched database. In addition, 952 protein clusters were identified using cluster analysis of the proteins identified by the peptides not unique to a single protein. This identification revealed that a minimum of 4,542 proteins could be identified from this experiment, representing ∼16% of all known mouse proteins. An evaluation of the number of false-positive identifications was undertaken by searching the entire MS/MS dataset against a database containing the sequences of over 12,000 proteins from archaea. This analysis allowed a systematic determination of the level of confidence in the identification of peptides as a function of SEQUEST cross correlation (Xcorr) and delta correlation (ΔCn) scores. Correlation charts were also constructed to show the number of unique peptides identified for proteins from specific classes. The results show that low-abundance proteins involved in signal transduction and transcription are generally identified by fewer peptides than high-abundance proteins that play a role in maintaining mammalian cellular structure and motility. The results presented here provide the broadest proteome coverage for a mammalian cell to date and show that MS-based proteomics has the potential to provide high coverage of the proteins expressed within a cell.

Targeting and Insertion of the Cholesterol-Binding Translocator Protein into the Outer Mitochondrial Membrane

Translocator protein (18 kDa, TSPO), previously known as the peripheral-type benzodiazepine receptor, is an outer mitochondrial membrane (OMM) protein necessary for cholesterol import and steroid production. We reconstituted the mitochondrial targeting and insertion of TSPO into the OMM to analyze the signals and mechanisms required for this process. Initial studies indicated the formation of a mitochondrial 66 kDa complex through Blue Native-PAGE analysis. The formation of this complex was found to be dependent on the presence of ATP and the cytosolic chaperone Hsp90. Through mutational analysis we identified two areas necessary for TSPO targeting, import, and function: amino acids 103-108 (Schellman motif), which provide the necessary structural orientation for import, and the cholesterol-binding C-terminus required for insertion. Although the translocase of the outer mitochondrial membrane (TOM) complex proteins Tom22 and Tom40 were present in the OMM, the TOM complex did not interact with TSPO. In search of proteins involved in TSPO import, we analyzed complexes known to interact with TSPO by mass spectrometry. Formation of the 66 kDa complex was found to be dependent on an identified protein, Metaxin 1, for formation and TSPO import. The level of import of TSPO into steroidogenic cell mitochondria was increased following treatment of the cells with cAMP. These findings suggest that the initial targeting of TSPO to mitochondria is dependent upon the presence of cytosolic chaperones interacting with the import receptor Tom70. The C-terminus plays an important role in targeting TSPO to mitochondria, whereas its import into the OMM is dependent upon the presence of the Schellman motif. Final integration of TSPO into the OMM occurs via its interaction with Metaxin 1. Import of TSPO into steroidogenic cell mitochondria is regulated by cAMP.

Proteomic analysis of extracellular matrix and vesicles.

The extracellular matrix (ECM) is the connective tissue component generated and secreted by cells to provide structural and functional support, while extracellular vesicles are distinct membrane-enclosed structures present outside of eucaryotic cells that carry out distinct biological functions. Different cell types release distinct populations of vesicles that fulfill various functions. Exosomes are vesicles commonly secreted by a variety of cells, whereas matrix vesicles (MVs) are specifically produced and secreted by bone cells to facilitate the formation of the ECM. This article focuses on the characteristics of the ECM and extracellular vesicles, and reviews the latest progress in applying proteomic technologies to analyze these features. The findings and implications in developmental biology, tumor biology, immunology, biomarker discovery, and vaccine research are also discussed.

Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates.

High-throughput proteomic studies on formalin-fixed, paraffin-embedded (FFPE) tissues have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications to proteins. We previously reported a method for the formation of ‘tissue surrogates’ as a model to study formalin fixation, histochemical processing, and protein retrieval from FFPE tissues. In this study, we demonstrate the use of high hydrostatic pressure as a method for efficient protein recovery from FFPE tissue surrogates. Reversal of formaldehyde-induced protein adducts and crosslinks was observed when lysozyme tissue surrogates were extracted at 45 000 psi and 80–100°C in Tris buffers containing 2% sodium dodecyl sulfate and 0.2 M glycine at pH 4. These conditions also produced peptides resulting from acid-catalyzed aspartic acid cleavage. Additives such as trimethylamine N-oxide or copper (II) chloride decreased the total percentage of these aspartic acid cleavage products, while maintaining efficient reversal of intermolecular crosslinks in the FFPE tissue surrogates. Mass spectrometry analysis of the recovered lysozyme yielded 70% sequence coverage, correctly identified all formaldehyde-reactive amino acids, and demonstrated hydrolysis at all of the expected trypsin cleavage sites. This study demonstrates that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues for proteomic analysis.

CD44 Promotes Intoxication by the Clostridial Iota- Family Toxins

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44+ melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.

NKX3.1 Homeodomain Protein Binds to Topoisomerase I and Enhances Its Activity

The prostate-specific homeodomain protein NKX3.1 is a tumor suppressor that is commonly down-regulated in human prostate cancer. Using an NKX3.1 affinity column, we isolated topoisomerase I (Topo I) from a PC-3 prostate cancer cell extract. Topo I is a class 1B DNA-resolving enzyme that is ubiquitously expressed in higher organisms and many prokaryotes. NKX3.1 interacts with Topo I to enhance formation of the Topo I-DNA complex and to increase Topo I cleavage of DNA. The two proteins interacted in affinity pull-down experiments in the presence of either DNase or RNase. The NKX3.1 homeodomain was essential, but not sufficient, for the interaction with Topo I. NKX3.1 binding to Topo I occurred independently of the Topo I NH2-terminal domain. The binding of equimolar amounts of Topo I to NKX3.1 caused displacement of NKX3.1 from its cognate DNA recognition sequence. Topo I activity in prostates of Nkx3.1+/- and Nkx3.1-/- mice was reduced compared with wild-type mice, whereas Topo I activity in livers, where no NKX3.1 is expressed, was independent of Nkx3.1 genotype. Endogenous Topo I and NKX3.1 could be coimmunoprecipitated from LNCaP cells, where NKX3.1 and Topo I were found to colocalize in the nucleus and comigrate within the nucleus in response to either gamma-irradiation or mitomycin C exposure, two DNA-damaging agents. This is the first report that a homeodomain protein can modify the activity of Topo I and may have implications for organ-specific DNA replication, transcription, or DNA repair.

Electrophoresis and liquid chromatography/tandem mass spectrometry in disease biomarker discovery

The search for disease markers is not new; however, with the emergence of new technologies such as nano-HPLC and electrospray ionization and time of flight mass spectrometry, the search has intensified considerably. Genomic, proteomic and metabolomic technologies are being used to search for novel disease markers. In this manuscript emphasis will be on different HPLC and MS methods that are used to search for metabolites and proteins that can be used for the discovery of novel, sensitive and specific disease biomarkers. Definitions of terms such as sensitivity, specificity, and protein profiles will be given. Methods used for effective fractionation, separation and quantitation of proteins and peptides using HPLC/MS will be discussed and examples are presented. A brief discussion of electrophoretic procedures used for protein fractionation and biomarker discovery is also included.

Proteomic biomarker discovery: It's more than just mass spectrometry

The previous decade witnessed an enormous number of studies with the singular goal of identifying protein biomarkers for diseases such as cancer. A large majority of these studies have focused on comparative studies of serum or plasma obtained from disease‐affected and control patients. In these studies, proteins identified in the samples using MS were compared with the hope that differences between samples would reveal useful biomarkers. Unfortunately, finding clinically relevant biomarkers has often been elusive and frustrating. As with most research efforts, both successes and failures, much has been learned about what strategies work and which do not. Part of the problem can be attributed to underestimating the effort required to discover novel biomarkers and depending too heavily on MS analysis of peripheral blood samples. Fortunately, the future for biomarker discovery still appears bright. MS technology continues to increase in sensitivity, throughput, and accuracy while novel types of samples and clever experimental designs coupled with innovative bioinformatics will make this vision of routine biomarker discovery a reality. To achieve ultimate success is going to require concomitant application of a number of different technologies, all providing the information necessary for discovering and validating clinically useful biomarkers.

Combined Blood/Tissue Analysis for Cancer Biomarker Discovery: Application to Renal Cell Carcinoma

A method that relies on subtractive tissue-directed shot-gun proteomics to identify tumor proteins in the blood of a patient newly diagnosed with cancer is described. To avoid analytical and statistical biases caused by physiologic variability of protein expression in the human population, this method was applied on clinical specimens obtained from a single patient diagnosed with nonmetastatic renal cell carcinoma (RCC). The proteomes extracted from tumor, normal adjacent tissue and preoperative plasma were analyzed using 2D-liquid chromatography-mass spectrometry (LC-MS). The lists of identified proteins were filtered to discover proteins that (i) were found in the tumor but not normal tissue, (ii) were identified in matching plasma, and (iii) whose spectral count was higher in tumor tissue than plasma. These filtering criteria resulted in identification of eight tumor proteins in the blood. Subsequent Western-blot analysis confirmed the presence of cadherin-5, cadherin-11, DEAD-box protein-23, and pyruvate kinase in the blood of the patient in the study as well as in the blood of four other patients diagnosed with RCC. These results demonstrate the utility of a combined blood/tissue analysis strategy that permits the detection of tumor proteins in the blood of a patient diagnosed with RCC.