Identification on membrane and characterization of phosphoproteins using an alkoxide-bridged dinuclear metal complex as a phosphate-binding tag molecule.

We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3.

Direct on-membrane peptide mass fingerprinting with MALDI-MS of tyrosine-phosphorylated proteins detected by immunostaining.

We have identified tyrosine-phosphorylated proteins on membrane from A-431 human epidermoid carcinoma cells by using detection with anti-phosphotyrosine antibody followed by PMF analysis. In there, on-membrane digestion for these protein spots was carried out on microscale region using chemical inkjet technology and the resulting tryptic digests were directly analyzed by MALDI-TOF MS. Proteins identified by a database search included phosphoproteins that are known to be markedly phosphorylated on tyrosine sites after the cells are treated with epidermal growth factor (EGF). This procedure is a rapid and easily handled approach that enables both detection and identification of phosphoproteins on a single blot membrane.

Development of Antibody-Drug Conjugates Using DDS and Molecular Imaging.

Antibody-drug conjugate (ADC), as a next generation of antibody therapeutics, is a combination of an antibody and a drug connected via a specialized linker. ADC has four action steps: systemic circulation, the enhanced permeability and retention (EPR) effect, penetration within the tumor tissue, and action on cells, such as through drug delivery system (DDS) drugs. An antibody with a size of about 10 nm has the same capacity for passive targeting as some DDS carriers, depending on the EPR effect. In addition, some antibodies are capable of active targeting. A linker is stable in the bloodstream but should release drugs efficiently in the tumor cells or their microenvironment. Thus, the linker technology is actually a typical controlled release technology in DDS. Here, we focused on molecular imaging. Fluorescent and positron emission tomography (PET) imaging is useful for the visualization and evaluation of antibody delivery in terms of passive and active targeting in the systemic circulation and in tumors. To evaluate the controlled release of the ADC in the targeted area, a mass spectrometry imaging (MSI) with a mass microscope, to visualize the drug released from ADC, was used. As a result, we succeeded in confirming the significant anti-tumor activity of anti-fibrin, or anti-tissue factor-ADC, in preclinical settings by using DDS and molecular imaging.

Imaging mass spectrometry for the precise design of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a class of immunotherapeutic agents that enable the delivery of cytotoxic drugs to target malignant cells. Because various cancers and tumour vascular endothelia strongly express anti-human tissue factor (TF), we prepared ADCs consisting of a TF-specific monoclonal antibody (mAb) linked to the anticancer agent (ACA) monomethyl auristatin E (MMAE) via a valine-citrulline (Val-Cit) linker (human TF ADC). Identifying the most efficient drug design in advance is difficult because ADCs have complicated structures. The best method of assessing ADCs is to examine their selectivity and efficiency in releasing and distributing the ACA within tumour tissue. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) can be used to directly detect the distributions of native molecules within tumour tissues. Here, MALDI-IMS enabled the identification of the intratumour distribution of MMAE released from the ADC. In conclusion, MALDI-IMS is a useful tool to assess ADCs and facilitate the optimization of ADC design.

Two-dimensional electrophoresis/phage panning (2D-PP): a novel technology for direct antibody selection on 2-D blots.

We describe a novel method, two-dimensional electrophoresis/phage panning (2D-PP), for the generation of antibodies against proteins in crude biochemical samples, such as cellular membrane fractions. These sources have traditionally presented problems as to the development of antibodies by conventional techniques. 2D-PP involves two-dimensional resolution of proteins, blotting of the proteins onto a nitrocellulose membrane, and screening of a phage antibody library and isolation of corresponding antibodies. By 2D-PP with detergent-insoluble "lipid rafts" as a target protein complex, we obtained specific phage pools against eight antigen spots (from a total of 39 spots). These antibodies were functional in Western blotting, enzyme-linked immunosorbent assaying (ELISA), and immunoscreening of a cDNA expression library. Propagation of anti-nitrocellulose phages was the major problem in 2D-PP, but was overcome by the use of the soluble anti-nitrocellulose antibody fragment. 2D-PP constitutes a key tool for functional analysis of proteins in complex fractions.

The significance of microscopic mass spectrometry with high resolution in the visualisation of drug distribution.

The visualisation and quantitative analysis of the native drug distribution in a pre-clinical or clinical setting are desirable for evaluating drug effects and optimising drug design. Here, using matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-IMS) with enhanced resolution and sensitivity, we compared the distribution of a paclitaxel (PTX)-incorporating micelle (NK105) with that of PTX alone after injection into tumour-bearing mice. We demonstrated optically and quantitatively that NK105 delivered more PTX to the tumour, including the centre of the tumour, while delivering less PTX to normal neural tissue, compared with injection with PTX alone. NK105 treatment yielded a greater antitumour effect and less neural toxicity in mice than did PTX treatment. The use of high-resolution MALDI-IMS may be an innovative approach for pharmacological evaluation and drug design support.

Novel in situ pretreatment method for significantly enhancing the signal in MALDI-TOF MS of formalin-fixed paraffin-embedded tissue sections.

The application of matrix-assisted laser desorption/ionization (MALDI)-based mass spectrometry (MS) to the proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue presents significant technical challenges. In situ enzymatic digestion is frequently used to unlock formalin-fixed tissues for analysis, but the results are often unsatisfactory. Here, we report a new, simplified in situ pretreatment method for preparing tissue sections for MS that involves heating with vapor containing acetonitrile in a small airtight pressurized space. The utility of the novel method is shown using FFPE tissue of human colon carcinoma. The number and intensity of MALDI peaks obtained from analysis of pretreated tissue was significantly higher than control tissue not subjected to pretreatment. A prominent peak (m/z 850) apparently specific to cancerous tissue was identified as a fragment of histone H2A in FFPE tissue pretreated using our method. This highly sensitive treatment may enable MALDI-MS analysis of archived pathological FFPE samples, thus leading to the identification of new biomarkers.

Direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric identification of proteins on membrane detected by Western blotting and lectin blotting.

We have developed new procedures to identify proteins after they are detected by Western blotting or other interactions such as lectin blotting on membranes. Our method is based on the combination of on-membrane MALDI-TOF mass spectrometry with piezoelectric chemical inkjet technology. Using this method the GroEL, FtsZ, DnaK, and GroES proteins were successfully identified from Escherichia coli after separation on two-dimensional gels, immunostaining, and on-membrane digestion. A glycoprotein detected by lectin blotting with concanavalin A was also identified using this technique.

Direct MS/MS analysis of proteins blotted on membranes by a matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight tandem mass spectrometer.

We constructed a system for the microscale identification of membrane-blotted proteins by proteolytic digestion using an instrument developed with piezoelectric chemical inkjet technology and MS/MS analyses of the resulting peptides with a matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight tandem mass spectrometer (MALDI-QIT-TOF MS). Using this system, bovine serum albumin was clearly identified at levels less than 100 fmol, and proteins from an Escherichia coli extract were also identified by an MS/MS ion search.

In situ phage screening. A method for identification of subnanogram tissue components in situ.

We have established a novel method, in situ phage screening (ISPS), to identify proteins in tissue microstructures. The method is based on the selection of repertoires of phage-displayed antibody fragments with small samples of tissues microdissected using a laser. Using a human muscle frozen section with an area of 4800 microm2 as a model target, we successfully selected monoclonal antibody fragments directed against three major (myosin heavy chain, actin, and tropomyosin-alpha) and one minor (alpha-actinin 2) muscle constituent proteins. These proteins were present in the sample in amounts less than one nanogram, and the antibodies were used to visualize the proteins in situ. This shows that the use of ISPS can obtain monoclonal antibodies for histochemical and biochemical purposes against minute amounts of proteins from microstructures with no requirement for large amounts of samples or biochemical efforts.