Phosphorylation is the most important protein post-translational modification, and affects almost all cellular processes. Phos-tag is a functional molecule that binds specifically phosphorylated ions. Technologies using Phos-tag are powerful tools for phosphoproteomics. Tip column chromatography using Phos-tag agarose is a superior method for the enrichment of phosphorylated peptides for mass spectrometric analysis. Phos-tag SDS-PAGE using Phos-tag acrylamide allows the specific separation of phosphorylated proteins by inducing an electrophoretic mobility shift. Using Phos-tag SDS-PAGE, we can obtain significant and abundant information about multiple phosphorylated forms of individual proteins. At present, however, the protocol for Phos-tag SDS-PAGE is not capable of analyzing multiple proteins. We expect to overcome this hurdle through further development of the technology. We believe that Phos-tag SDS-PAGE has enormous potential for accelerating the progress of proteomics research using electrophoresis.

Tau is a microtubule-associated protein primarily expressed in the axons of neurons. Tau regulates microtubule dynamics and transport of organelles along microtubules in axons. These functions of tau are regulated by phosphorylation with a number of protein kinases. Tau is also a major component of neurofibrillary tangles (NFTs) in the brains of patients with tauopathies, including Alzheimer’s disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Tau in NFTs is hyperphosphorylated, and therefore, the phosphorylation of tau has been intensively investigated. To date, more than 40 phosphorylation sites have been identified in aggregated tau. However, the physiological and pathological roles of the respective phosphorylation sites and how their phosphorylation is regulated are unclear. This could be due to the lack of the proper method of analyzing tau phosphorylation. Most of previous phosphorylation studies were conducted with a repertory of phosphorylation-site specific antibodies. While they provide relative changes of respective phosphorylation sites, it is hard to estimate total or absolute phosphorylation. To overcome these problems, we have recently applied the Phos-tag technique to the analysis of tau phosphorylation. This method separated tau into many bands on SDS-PAGE depending on phosphorylation, making it possible to analyze tau phosphorylation quantitatively and combinatorially. We identified phosphor-isotypes of tau expressed in cultured cells and found the abundance of nonphosphorylated tau in mouse and human tauopathy brains. Our results demonstrate that the Phos-tag would be a powerful method to characterize in vivo physiological and pathological phosphorylation of tau.

Protein phosphorylation is one of the most widespread types of post-translational modifications in eukaryotes and can regulate diverse properties of proteins. Protein kinases are encoded by over 500 genes in the human genome and play critical roles in various signaling pathways. In fact, many diseases are associated with mutations in protein kinases. To fully and therapeutically understand the complex signaling networks, it is essential to develop analytical strategies for the global identification and functional characterization of in vivo substrates of individual protein kinases. Recent advances in various phosphoproteomic technologies such as Phos-tag Western blotting have enabled efficient and detailed analysis of protein kinase substrates. Here, we describe cellular functions of the newly identified substrates of three disease-associated protein kinases including ERK, PKD and PINK1.

Autophagy is an intracellular recycling system conserved in eukaryotic cells. During autophagy, a part of cytoplasm is sequestered by a double-membrane structure, called the autophagosome, and transported to a lytic compartment to be degraded. At the initial step of autophagosome formation, Atg13 functions as a multipartite regulatory hub responsible for supramolecular assembly of the Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 complex); Atg13 consists of two regions, the N-terminal HORMA domain and the C-terminal intrinsically disordered region (IDR), and possesses an Atg1-binding region and two distinct Atg17-binding regions in the IDR, in which more than 40 serine residues are phosphorylated under nutrient-rich conditions. In response to starvation, these residues are dephosphorylated and Atg13 enhances the interactions with Atg1 and two molecules of Atg17, which leads to sequential assembly of the Atg1 complexes.

Protein phosphorylation is a key post-translational modification for regulation of the signal transduction in the cells. Recent reports have indicated that the almost proteins in the cells are phosphorylated. In general, a specific antibody recognizing its phosphorylation site is used to analyze protein phosphoarylation. However, generation of the specific anti-phospho-site antibody requires a very hard work. Phos-tag developed by Kinoshita group in Hiroshima University, functions as a specific phosphorylation binder. In this study, we demonstrate two detection methods using Phos-tag: 1) autophosphorylation activity of protein kinases by biotinylated Phos-tag with AlphaScreen technology and 2) phosphorylated protein in the cell lysates using Phos-tag acrylamide SDS-PAGE. These results indicated that Phos-tag is a very useful tool for biochemical and cell biological analyses of protein phosphorylation.

Exosomes are defined as lipid bilayered extracellular vesicles with 40–100 nm diameter. Their molecular characteristics and biological behaviors are now extensively investigated in areas of cancer diagnosis and DDS development, because they copy cancer cell-derived molecules (nucleic acids, proteins, and metabolites) and paste them to distant recipient cells. Recently we developed a couple of key technologies; Exosome immuno-capture tip (anti-CD9 MSIA tip) and EV-Second (Extracellular Vesicle Isolation by Size Exclusion Chromatography on Drip Column, GL Sciences International), allowing rapid isolation of high quality exosomes from serum/plasma or cell culture medium by simple procedures. Including these new tools, technological features of each exosome purification method are demonstrated with practical examples. Using these devices, proteome-wide screening of early detection biomarkers for lung cancer or gastric cancer. In addition, it was the first report showing detection of a major virulence factor of Helicobacter pylori CagA within exosomes purified from sera of patients infected with H. pylori. We further confirmed “functional” delivery of CagA proteins from H. pylori-infected cells to uninfected cells via exosomes, suggesting that CagA-containing exosomes may be involved in development of extragastric disorders associated with H. pylori infection.

Serum/plasma contains thousands of different types of proteins and peptides that can provide valuable information on numerous inherent processes. However, the existence of high-abundance proteins and the large dynamic range of serum proteins/peptides make analysis of low-abundance proteins/peptides challenging. In particular, detailed analysis and identification of native peptides remain extremely difficult due to the tremendous variety of cleavage possibilities and posttranslational modifications. Therefore, widely ranging searches based on peptide identification are difficult. Herein, I describe the highly accurate and sensitive quantitative analysis of over 2,500 peptides. The strategy combined isobaric tag labeling, amine-reactive 6-plex tandem mass tag labeling, and a modified differential solubilization method for high-yield peptide extraction. We quantitatively analyzed six pooled plasma samples (three pre-surgery and three post-surgery) to discover potential candidate biomarker peptides of renal cell carcinoma. The concentrations of about 30 peptides were found to be altered following surgery. A preliminary validation study was conducted using 77 plasma samples to demonstrate the possibility that even unidentified potential candidate biomarker peptides can be verified using the dimethyl labeling method.

A serum protein fraction is employed as a screening for serum protein abnormalities widely and is essential for diagnosis and treatment of multiple myeloma through a detection of monoclonal protein in particular. The biggest significance, for which we conduct serum protein fraction in the hospital laboratory, is will be to let us to medical doctors an important view immediately. It is because we can read a patient’s case record with abnormal finding and consider the doubted clinical conditions. For example, characteristic β–γ bridging can suggest IgG4 related disease, it’s possible to consult other views and doubt IgG4 high price. When admitting nonspecific reactions biochemical and immunochemical tests, we can check the influence of monoclonal proteins. It is also useful for judgment of cryoglobulin type.

Temperature-dependent proteins are abnormal immunoglobulins, which exhibit varying properties with variation in temperature. Cryoglobulins are abnormal immunoglobulins that precipitate at cold temperature. Some types of cryoglobulins cause Raynaud’s phenomenon which develops severe damage of blood vessel in extremities by cold exposure. The proteins are also involved in nephropathy and hyperviscous syndrome of unknown origin. Cryoglobulins are divided into three types by the constituents of immunoglobulins: (1) type I, which has a monoclonal (M) protein only; (2) type II, which has an M protein and a polyclonal immunoglobulin (Ig); and (3) type III, which has an only polyclonal Ig. It is remarkably difficult to precisely distinguish among these variations because there are numerous factors that prevent clear observation of the M protein for cryoglobulin types II and III. In addition, this type of immunoglobulin is largely observed in a range of its standard values. Nonetheless, it has been confirmed that even a small amount of M protein can cause severe symptoms in patients. Therefore, it is useful to identify and analyze the specific type of cryoglobulin involved in the symptoms of a particular patient, and give the type as an additional comment to doctor in charge of the patient. We hope that the comment will provide incremental therapeutic value beyond the report of the clinical investigation itself.

SDS-agarose gel electrophoresis coupled with an acid violet staining has been used for classification of urinary protein patterns of renal diseases. The electrophoresis is easy to operate with better resolution of protein separation based on molecular masses. In this study, we examined suitable protocols of silver staining and western blotting for the electrophoretic gels. As a result, protein bands on SDS-agarose gel were visualized with the colloidal silver staining. When zinc sulfate was added to the protein fixing solution, the silver staining was ~30-fold more sensitive to the detection of protein bands than the acid violet staining. Furthermore, gravity blotting was effective for transferring proteins separated by SDS-agarose gels to PVDF membranes to detect specific proteins by western blotting. These results suggest that SDS-agarose gel electrophoresis has the added benefit of being potentially used for fundamental biochemical research.

Multiple myeloma (MM) is a refractory plasma cell tumor. It is a functional tumor exhibiting various molecular pathologies, including the secretion of monoclonal immunoglobulin, detected as M protein by cellulose acetate membrane electrophoresis. Recent advances in myeloma research have led to progress in the specific analysis of this disease in patients. Evolution in the diagnosis and treatment of MM has contributed to improved prognosis in patients with this disease. Research on MM has therefore become a remarkable example of translational cancer research. This paper, entitled “Advances in clinical and basic research on multiple myeloma based on electrophoresis methods,” introduces Session 3 of the 68^th annual meeting of the Japanese Electrophoresis Society. This paper describes the recent acceleration in translational research on MM, as well as outlines the relationship between in vitro diagnostic methods and new treatments of this disease.

Three factors: the identification of myeloma cells by surface antigen analysis, the ability to measure serum free light chains, and the advancement of elucidation of chromosomes and genetic abnormalities have led to the advancement of diagnosis, monitoring, risk classification and response criteria in multiple myeloma. Many molecular targeted drugs have been introduced in treatment, and remarkable prognostic improvement has been obtained, but high-dose chemotherapy, which is particularly effective for immature type myeloma patients, is useful even in the era of new agents. The goal of treatment is now changing to the disappearance of minimal residual disease because getting a deeper remission directly affects the prolongation of the survival rate. The road to the cure of multiple myeloma may finally be opened.

Disease progression is a major obstacle to achieving cure in patients with multiple myeloma (MM). Recent clinical studies suggest the contribution of infections to MM progression; however, the underlying mechanisms remain unexplained. In our cohort of MM patients, we found that disease progression frequently occurred after bacterial infections. The CD180/MD-1 complex, one of LPS receptors, was specifically expressed on MM cells and its abundance was markedly up-regulated under adherent and hypoxic conditions. Bacterial LPS enhanced the growth of MM cells in positive correlation with the expression levels of CD180 in vitro and in vivo. We identified IKZF1 (Ikaros) as a pivotal transcriptional activator of the CD180 gene. Accordingly, genetic and pharmacological targeting of Ikaros ameliorated the response of MM cells to LPS in vitro and in vivo. Consistent with these findings, infection-triggered progression was not observed in MM patients under treatment with Ikaros-degrading immunomodulatory drugs. The LPS-CD180/MD-1 pathway represents a novel mechanism of disease progression and is a therapeutic target for prolonging survival in MM patients.

We established B cell derived induced pluripotent stem cells (BiPSCs) with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes. The critical points of our method are pre-stimulation of B cells with IL-21 and CD40-ligand (CD40L), followed by consecutive transfection of highly concentrated Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) using a retroviral vector. Following each transfection the cells were centrifuged onto a retronectin coated plate and the activated by IL-4, IL-2, and CD40L. Furthermore, we established BiPSCs in which activation-induced cytidine deaminase (AID) could be induced using the doxycycline-controlled (BiPSC-A). Both the parental BiPSC and BiPSC-A showed the capability of differentiating into hematopoietic progenitor cells (HPCs) based on confirming CD34 expression and colony-formation of macrophage, granulocyte, and erythrocyte from CD34-positive cells. The findings that BiPSC-A can differentiate into HPCs suggest that induction of AID expression would result in chromosomal translocations in the progress of differentiation from BiPSCs. Therefore, that these BiPSCs could be useful in elucidating the tumor origin of abnormal B cells in myelomagenesis.

Mitochondria are two-membrane bounded organelles consisting of over 1000 different proteins, most of which are synthesized on the cytosolic ribosomes and subsequently imported into mitochondria. The TOM40 complex, one of the major mitochondrial protein translocators, functions as an entry gate for most mitochondrial precursor proteins. The central subunit Tom40, which provides a pore for protein translocation by a β-barrel structure, is also imported into mitochondria. Accumulating evidence has shown that the assembly of the Tom40 precursor into the final TOM40 complex occurs through several steps. Here we review the current view on the roles and assembly processes of mitochondrial translocator complexes revealed by Blue-Native PAGE combined with in vitro mitochondrial protein import assay.

Mitochondria are essential cellular organelles that function as a major power supply for the cell. Their dysfunction result in prototypic mitochondrial diseases, which mainly exhibit muscle weakness and central nervous system abnormalities, and also in various pathological states such as aging, diabetes and cancer. Recent studies based on blue native polyacrylamide gel electrophoresis (BN-PAGE) have clarified that the mitochondrial respiratory chain complexes (complexes I-IV) further form their supercomplex structure in mitochondrial inner membrane. The respiratory supercomplex assembly and its stability may closely relate to the efficiency of mitochondrial respiration and energy production. We discovered that cytochrome c oxidase subunit 7a related polypeptide (COX7RP), also known as COX7A2L/SCAF1, is the first identified factor that promotes supercomplex assembly. Gain- and loss-of-function studies for COX7RP demonstrated that the molecule plays a critical role in muscle endurance and adaptive thermogenesis in vivo. As exemplified by our COX7RP studies, BN-PAGE is a powerful technique for the analysis of mitochondrial supercomplex assembly and will further dissect mitochondrial dysfunction that contributes to disease etiology.

Blue native (BN) PAGE, in which proteins are charged with the anionic dye CBB G-250, has been widely used to resolve protein complexes. We applied this technique to analyse assembly and disassembly processes of cyanobacterial Kai protein complexes in an in-vitro circadian clock reconstitution system. The circadian clock is an intracellular timing mechanism in living organisms that coordinates their metabolisms with diurnal environmental changes. Cyanobacteria are the simplest organisms showing circadian rhythms. Self-sustainable oscillation of KaiC phosphorylation has been reconstituted in vitro by mixing three clock proteins, KaiA, KaiB and KaiC, with ATP, demonstrating that this cycle is the basic time generator of the circadian clock of cyanobacteria. Here, we tracked the assembly and disassembly processes of the three Kai proteins over time using BN-PAGE. When KaiC proteins were separated in BN-PAGE, they were resolved as two bands corresponding to a KaiC hexamer and a KaiC monomer. However, when KaiC was analysed by gel filtration chromatography, no KaiC monomer was observed. This discrepancy was explained by the effect of CBB on KaiC hexamer, which was confirmed by a CBB-free native PAGE. These data indicated two conformational states of KaiC hexamer and show that the ground-state KaiC (gs-KaiC) is stable enough during BN-PAGE, and competent-state KaiC (cs-KaiC) is labile and disassembled into monomers in the presence of CBB.

Blue-Native (BN)-PAGE is an electrophoresis for separating protein complexes while maintaining the structure and function. BN-PAGE has been widely used for analysis of protein complexes due to its simplicity and applicability, and thus we have also used it for various analyses. Here, we briefly introduce these analyses and especially focus on the application for the comprehensive analysis of protein complexes, which was rather difficult from the viewpoint of time and cost so far.

Milk contains two kinds of secretory membrane microvesicles, milk fat globule and exsosome. Using SDS-PAGE in combination with density gradient ultracentrifugation, a phosphatidyl serine (PS)-binding membrane protein, MFG-E8, of these membrane vesicles in mouse milk was investigated. Milk fat globules and exosomes in fresh milk from lactating mice were recovered mainly from the lower density (~1.0) fractions and higher density (1.07~1.13) fractions, respectively, and MFG-E8 distributed to the milk fat globule fractions. Such MFG-E8 distribution was changed after forced weaning (pups withdrawal), i.e., MFG-E8 was present in not only the milk fat globule fractions but also a wide density-range (1.0~1.1) of the fractions including the exosome fractions and soluble protein fractions. Fluorescence microscopy analysis of the milk fat globules using anti-MFG-E8 antibody and annexin V as a PS probe showed that both of MFG-E8 and PS on the surface of milk fat globules largely differed even in fresh milk, and increased both in vivo with time after weaning and in vitro during the milk incubation. Mammary epithelial cells are known as non-professional phagocytes, which play a role in clearance of neighboring apoptotic cells on the epithelial cell turnover in lactating mammary glands. Mammary epithelial HC-11 cells preferentially phagocytosed old milk-fat globules rich in the surface MFG-E8 and, moreover, such phagocytosis was inhibited by anti-MFG-E8 antibody. We hypothesize that mammary epithelial cells may remove aged milk fat globules labeled heavily with MFG-E8 from breast milk and maintain freshness of milk lipids as an important nutrient for nursing infants.

To develop novel sero-diagnostic markers for membranous proteins of lung cancer, surface proteins were identified in combined with various proteomic methods. Biotinylated membranous proteins in lung cancer cell lines were collected and identified by the shot-gun analysis. Expression levels of identified proteins in four different histological types of lung cancer cell line were examined by the immunoblot and immunohistochemical analyses. Serum levels of these proteins were also examined in patients with lung cancer and healthy controls by the reverse-phase protein array (RPPA) method. From identified 92 membranous proteins including CD109, CD155 and Roundabout Guidance Receptor 1 (ROBO1), we focused on the CD155 protein. Serum levels of CD155 were significantly higher in patients with adenocarcinoma and squamous cell carcinoma than in healthy controls (p < 0.0001), and the area under the curve (AUC) of receiver-operating characteristic curve (ROC) analysis was 0.87, when an optimal cut-off value of 0.31 was applied, the diagnostic sensitivity and specificity for lung cancer were 81 and 82%, respectively. To our knowledge, this is the first report providing evidence that CD155 may be a sero-diagnostic marker for non-small cell lung cancer. This methodology was shown to be useful for acquisition of novel biomarkers for membranous proteins.

Here, we review the utility of immunohistochemical (IHC) staining as a diagnostic assay in cancers. IHC staining for hormone receptors is used to select patients for hormonal therapy in breast cancers. IHC staining and fluorescent in situ hybridization (FISH) are well-established assays for overexpression of human epidermal growth factor receptor 2 (HER2) protein and amplification of the HER2 gene, respectively; positive results indicate that targeted therapy with trastuzumab, the anti-HER2 antibody, is appropriate. Similarly, the anaplastic lymphoma kinase (ALK) inhibitors, crizotinib and alectinib, can treat non-small cell lung cancers with ALK fusion genes, which are found through diagnostic assays that include IHC staining and FISH; and imatinib is effective for KIT-expressing gastrointestinal stromal tumors. IHC confirmation of target protein expression is also necessary before using targeted agents in hematologic malignancies: CD20 for rituximab, CD30 for brentuximab vedotin, and CC chemokine receptor-4 for mogamurizumab. IHC staining for programmed death-ligand 1 is used to identify non-small cell lung cancer patients who can respond to pembrolizumab, which targets programmed death-1. This review also discusses possible prediction of response to cytotoxic agents through IHC staining for solute carrier transporters, and the differences between IHC staining and western blotting.

We have generated several tumor markers for lung cancer using various proteomic methods including generation of monoclonal antibodies by using the random immunization method, the two-dimensional electrophoresis, and autoantibody analysis using lung cancer tissues, cell lines and patients’ sera. Although the adjuvant platinum-based chemotherapy has been shown to improve survival of patients with completely resected stage II and IIIA non-small cell lung cancer (NSCLC), its effect is limited. In addition, the prior clinical examination whether this therapy is effective has not been performed. In the present study, we investigated the utility as the therapeutic effect prediction markers about the candidate marker proteins for lung cancer that we obtained previously. We review mainly on our recent study about the utility of nestin, S100A16, and myosin-9 proteins as the effect prediction factor for the adjuvant platinum-based chemotherapy.

Myxofibrosarcoma (MFS) is a mesenchymal malignancy characterized by frequent recurrence even after radical wide resection. To optimize therapy for MFS patients, we aimed to identify candidate tissue biomarkers of MFS invasion potential. We performed protein profiling in invasive and non-invasive MFS and detected a number of differentially expressed proteins. Among them, DCBLD2 was identified as a novel factor associated with tumor cell invasion in MFS, suggesting its potential clinical application as a biomarker for MFS prognosis. This is the first report about possible clinical utility of DCBLD2 in sarcomas. Further investigation of the detected proteins may produce additional biomarkers or clinical targets in MFS.

Rare cancers are defined as malignancies with an incidence of fewer than 6 per 100,000 persons per year. There are approximately 200 different types of cancers which are identified as rare cancer, and they account for approximately 20% of all newly-diagnosed cancer patients. As the number of cases of each rare cancer is quite limited, the clinical study is unusually difficult, and the use of biomarkers for patient stratification is especially important. To obtain the medical seeds for novel therapy for typical rare cancers, sarcomas, we have conducted cancer research. The re-location of anti-cancer drugs is a practical approach to novel therapeutic strategies for rare cancers. With this notion, we developed patient-derived cancer models of sarcomas, and screened the growth inhibitory effects of anti-cancer drugs. The drugs used in our study were approved for the treatments of other malignancies. We found that there are anti-cancer drugs which exhibited anti-tumor effects for sarcoma cells with a considerably low concentration. Those anti-cancer drugs can be candidate drugs for sarcoma patients. The proteins as candidates of predictive biomarkers were also identified by proteomic approach. We consider that the use of patient-derived cancer models as well as proteomic approach are quite important approach to develop novel therapeutic strategy of rare cancers.

In a clinical diagnostic microbiology laboratory, the current method of identifying bacterial isolates is based mainly on phenotypic characteristics, such as the growth pattern on different media, colony morphology, Gram stain, and various biochemical reactions. These techniques collectively allow high-level accuracy in identifying most bacterial isolates, but they are costly and time-consuming. In our clinical microbiology laboratory, we prospectively assessed the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify bacterial strains that were routinely isolated from clinical samples. MALDI-TOF MS is a rapid, simple, and high-throughput proteomic technique for the identification of a variety of bacterial species. Since colony to colony differences and the effects of culture duration on the results are minimal, it can be implemented in a conventional laboratory setting. Although for some pathogens, the pre-analytic processes should be refined and current database should be improved to obtain more accurate results, the MALDI-TOF MS based method generally performs as well as the conventional methods and is a promising technology in clinical laboratories.

In recent years, a method using MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry) is beginning to be used as a new rapid microorganism identification method. Since operation is very simple, no special skills are required, and identification results are obtained rapidly, it is being introduced to food and beverage manufacturing companies in Japan. Therefore, we confirmed the possibility of identification and accuracy by using the genus Alicyclobacillus strains, thermophilic acidophilic bacterium, Genus Moorella starins and Genus Thermoanaerobacter strains, thermophilic anaerobis spore forming bacterium, which needs to be controlled in the soft drink manufacturing. In existing databases, the amount of data of these strains can be extremely small and cannot be identified. However, by constructing its own database, it was found that it is possible to identify with the same accuracy as the conventional method using 16S rDNA.

Microbiological monitoring, defined as the practice of scheduled, repetitive testing of animal colony for evidence of selected microbiological infections, is only way to guarantee the microbiological quality of laboratory animals. Our center introduced MALDI Biotyper for rapid identification of bacteria in 2013 for microbiological monitoring. However, default library of MALDI Biotyper was insufficient for pathogenic microorganisms in laboratory animals. Therefore, we have created a new library. In addition to microbiological monitoring of laboratory animals, we started contract testing of bacteria identification from various derivation like as ingredients of using MALDI Biotyper since 2013. We introduce the preparation of library of pathogenic microorganisms of laboratory animals and current status of request for microorganism identification test in this article.

In recent years, a technique using a mass spectrometer has attracted a great deal of attention as a new identification method of microorganisms, and around 2300 systems (MALDI Biotyper: Bruker) are introduced in various fields including clinical area around the world. This method is said to be “innovative technology” in clinical microbiology, because it can quickly obtain reliability almost equivalent to the identification result by 16S rRNA analysis. Now that MALDI-TOF MS is widely recognized as a microorganism identification system, the most needed in this field is application to drug resistance detection and typing. Then, we show the latest information and future possibility of how mass spectrometry technology is applied to these applications.

Identification of plant pathogenic microbe, plant virus, and small pest including spider mite species are still performed using morphological information, although DNA and other biological approaches have been attempted for discrimination purposes. These techniques need much time, are expensive, and require specialists. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, which has been used for the clinical investigation, is applied for rapid identification of plant pathogenic microbe, plant virus, and small pest.