Rapid progress has been made in identifying cellular phosphoproteins by shotgun phosphoproteomics using nanoLC-MS combined with phosphopeptide enrichment techniques. Because samples for shotgun proteomics exhibit extremely high complexity with a wide dynamic range of concentration, fractionation approaches before/after phosphopeptide enrichment prior to nanoLC-MS are often necessary to widen the phosphoproteome coverage. Recently, by using one-dimensional nanoLCMS with meter-long, monolithic silica-C18 capillary column, we successfully identified the proteome expressed in E. coli cells [Iwasaki et al, Anal Chem 2010] and human induced pluripotent stem cells [Yamana et al, JPR 2013]. We also developed a highly efficient phosphopeptide enrichment protocol based on aliphatic hydroxyl acid-modified metal oxide chromatography (HAMMOC) using lactic acid-modified titania [Sugiyama et al, MCP 2007]. We combined the‘one-shot’approach with HAMMOC, and applied to STY phosphoproteomics without any fractionation, identifying more than 12,000 phosphosites (3,700 phosphoproteins) from 0.25 mg HeLa lysate. We also employed sequential enrichment of tyrosine phosphopeptides using lactic acid-modified titania chromatography followed by immunoprecipitation using pY antibody cocktails. Furthermore, we minimized the required samples down to 10,000 cells (approx. 1 microgram of proteins) by miniaturizing LC, skipping autosampler and optimizing protocol based on phase-transfer surfactants [Masuda et al, JPR 2008], resulting in identification of more than 1,000 phosphopeptides [Masuda et al, Anal Chem 2011]. Based on these approaches, phosphorylation dynamics induced by kinase inhibitors were quantitatively analyzed to elucidate the mechanism of actions of these drugs [Imami et al, MCP 2012].

抄録全体を表示

Proteomic technologies allow a comprehensive study of multi-protein complexes that carry out many cellular functions in a higher-order network in the cell. The use of tagged proteins as affinity bait, coupled with mass spectrometric identification, enables us to isolate almost any functional protein complex or its synthetic intermediates that might represent snapshots of nascent functional protein complexes at particular stages of its biogenesis and to identify their constituents-some of which show dynamic changes for association with the intermediates at various stages of the biogenesis. The idea behind this snapshot analysis is that some of the associated proteins in one initially isolated complex can also be present in other precursor complexes, and thus would allow the purification of intermediates from different stages of biogenesis. Initially, we had applied this approach to the analysis of protein constituents of pre-ribosomes, and is now expanded this to the analysis of not only protein but also RNA constituents of small nuclear ribonucleoprotein (snRNP) intermediates formed during spliceosome biogenesis in cooperation with Dr. Isobe of Tokyo Metropolitan University. In this presentation, I would like to talk about mainly current status of the snapshot analysis of snRNP complexes.

抄録全体を表示

One methodology for biomarker discovery of chronic diseases exploits the fact that glycoproteins produced by impaired cells have altered glycan structures although the proteins themselves are common, ubiquitous, abundant and familiar. Here, we describe our strategy to approach the detection of these low-level glycoproteins as serum biomarkers: 1) A quantitative real-time PCR array for glycogenes to predict the glycan structures of secreted glycoproteins;2) Analysis by lectin microarray to select lectins which distinguish diseaserelated glycan structures on secreted glycoproteins; 3) An isotope-coded glycosylation-site-specific tagging (IGOT) high-throughput method to identify carrier proteins having the specific lectin epitope. 4) Selection of candidate molecules by skillful bioinformatics technology. 5) Re-selection of the best lectin distinguishing the glycan-alteration in the patient serum. 6) Final verification using more than 100 patient sera. Using this strategy, we have identified many glycoproteins containing glycan structures altered in impaired cells. These candidate glycoproteins were immunoprecipitated from serum using commercially available antibodies, and their glycan alteration was examined by a lectin microarray. Finally they were analyzed by multistage tandem mass spectrometry (MSⁿ). According to this protocol, we succeeded in establishment of a diagnosis kit for liver fibrosis. A novel marker hyperglycosylated Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA⁺-M2BP) was developed for liver fibrosis using the glycan “sugar chain” -based immunoassay. This kit was named FastLec-Hepa, and automatically detects unique fibrosis-related glyco-alteration in serum M2BP within 20 min. FastLec-Hepa is the only assay currently available for clinically beneficial therapy evaluation through quantitation of disease severity.

抄録全体を表示

Post-translational modifications (PTMs) frequently play a key role in regulating protein function. The proteasome is composed of at least 33 different subunits and is a multi-catalytic protease complex that degrades ubiquitinated proteins in eukaryotic cells. The comprehensive PTM analysis of proteasome subunits using proteomic techniques indicated that 21, 1, 1 and 28 subunits were N-acetylated, N-myristoylated, N-methylated and phosphorylated, respectively. Furthermore, the study using the yeast mutants showed that N-acetylation might be involved in the chymotrypsin-like activity and accumulation level of the 20S proteasome and that N-methylation of Rpt1 might be essential for cell growth or stress tolerance in yeast. Like this, it is evident that the PTMs significantly affect the function of the proteasome. The phosphorylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is also thought to play an important role in cell regulation and signal transduction. However, the relationship between hnRNP K phosphorylation and cellular events has only been indirectly examined, and the phosphorylated forms of endogenous hnRNP K have not been biochemically characterized in detail. The PTM analysis using 2-D Phos-tag affinity electrophoresis was successful to characterize multiple forms of hnRNP K produced by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. Furthermore, this analysis demonstrated that each form of hnRNP K was differentially modulated in response to external stimulation with bacterial lipopolysaccharide or serum. Like this, the PTM analysis is also crucial for a better understanding of the functional properties of proteins.

抄録全体を表示

Protein glycosylation is one of the most complicated post transcriptional modifications, while it plays diverse physiological functions by controlling protein folding, charge state, ligand-receptor interactions, or immunogenicity. To date, we have focused on developing novel glycoproteomic technologies which allowed rapid and comprehensive profiling of clinically-important glycan structure disorders on both targeted glycoproteins and even whole serum/plasma glycoproteins. Isotopic glycosidase elution and labeling on lectin-column chromatography (IGEL) technology (Mol Cell Proteomics, 2010, 9(9):1819) enabled us not only to identify N-glycosylation sites comprehensively but also to compare glycan structures on each glycosylation site quantitatively in a single LC/MS/MS analysis. Using this technology, we revealed that glycans on A2GL_Asn151,A2GL_Asn290, CD14_Asn132, CO8A_Asn417, C163A_Asn64, TIMP1_ Asn30, and TSP1_Asn1049 demonstrated lung cancer-specific alterations. Recently we developed Energy resolved oxonium ion monitoring (Erexim) technology to evaluate glycan microheterogeneities on therapeutic protein drugs (Anal Chem, 2012, 84(22):9655). The Erexim method can quantify ~50 glycan structure variations on a therapeutic antibody molecule according to energy-resolved MRM for oligosaccharide-derived oxonium ions in only 10 minutes. We discovered significant lot-to-lot variations of glycosylation profiles on Herceptin and Avastin. The existence of anaphylaxis-inducible glycan structures on Erbitux were also quantitatively determined by Erexim. This technology has already been put to practical use in R&D and CMC areas by a contract research organization (CRO). Thus I’d like to continue facilitating the life innovation by creation of further sophisticated MS-based glycoproteomic technologies in the future.

抄録全体を表示

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens responsible for nosocomial infection. The presence of MRSA in a hospital is very detrimental to patients and to hospital management. Thus, rapid identification of MRSA is needed. This study performed a prospective study of rapid discrimination of MSSA from MRSA using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. We evaluated 305 clinical isolates of S. aureus using the MALDI-TOF MS system and support vector machine. The predictive model was trained using 100 S. aureus isolates (50 MSSA and 50 MRSA). The identification rates were 90.2% for MSSA and 78.6% for MRSA following the 10-fold crossvalidation SVM. In the blind test sets, 205 S. aureus isolates (95 MSSA and 110 MRSA) were correctly classified. The identification rates were 95.8% for MSSA and 81.8% for MRSA. In conclusion, the method proposed in this study using a predictive model enables detection from one colony in 5 minutes, and thus is useful at clinical sites at which rapid discrimination of MRSA from MSSA is required.

抄録全体を表示

Medical ProteoScope (MPS) is the company of proteomics excellence towards the better quality of life. In clinical practice, there is a need for biomarkers to help improve treatment outcomes. Based on integration of the state-ofthe-art proteomics technology, we have focused on development of protein biomarkers. Label-free comparative LC-MS/MS is our core analytical platform, associated with the self-made algorithms, i-OPAL and i-RUBY, for data alignment of non-linearly fluctuated LC elution time. Under quality control (QC) operations of these technologies, collaborative studies with academic institutions and drug companies led to identification of proteins related closely to drug response, current state of the disease, progression of the disease and metastatic risk in the case of cancer patients. Posttranslational modifications (PTMs) of proteins are the promising targets for understanding disease mechanisms. Since 2008, MPS participates in the coordination funds presided by Yokohama City University. We developed differential analysis systems of PTMs including reversible phosphorylation. Formalin-fixed paraffinembedded (FFPE) tissues archived in hospitals have a potential for discovery of the therapeutic targets as well as proteomic biomarkers. We have optimized protocols to dissect minute lesions from the FFPE tissues. Proteomic data of the collected specimens enabled to evaluate more accurately the disease state of the tissues. Thus, as a proteomics leading venture, MPS is continuously contributing to the fields of medicine.

抄録全体を表示

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing lowly abundant protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a subsequently linked high-performance mass spectrometer system. The hollow-fiber membrane based serum pre-treatment device we developed can efficiently deplete high-molecular-weight proteins and concentrate low-molecular-weight proteins/peptides automatically within an hour. The proteomic analysis of healthy human serum pre-treated using the device, followed by the mass spectrometer, successfully identified about 2,000 proteins. According to the concept of the pre-treatment device, 64% of the identified proteins were smaller proteins than the human serum albumin. We believe this unique serum pre-treatment device and the proteomic analysis protocol allow high-throughput and efficient discovery of serum disease biomarkers.

抄録全体を表示