Biomedical subjects

Tomoyo Nakano

Publications and source records attributed to Tomoyo Nakano.

4 recordsLinked to original sources

PubMed2005

Clinical-scale high-throughput human plasma proteome analysis: lung adenocarcinoma.

Clinical proteomics requires the stable and reproducible analysis of a large number of human samples. We report a high-throughput comprehensive protein profiling system comprising a fully automated, on-line, two-dimensional microflow liquid chromatography/tandem mass spectrometry (2-D microLC-MS/MS) system for use in clinical proteomics. A linear ion-trap mass spectrometer (ITMS) also known as a 2-D ITMS instrument, which is characterized by high scan speed, was incorporated into the microLC-MS/MS system in order to obtain highly improved sensitivity and resolution in MS/MS acquisition. This system was used to evaluate bovine serum albumin and human 26S proteasome. Application of these high-throughput microLC conditions and the 2-D ITMS resulted in a 10-fold increase in sensitivity in protein identification. Additionally, peptide fragments from the 26S proteasome were identified three-fold more efficiently than by the conventional 3-D ITMS instrument. In this study, the 2-D microLC-MS/MS system that uses linear 2-D ITMS has been applied for the plasma proteome analysis of a few samples from healthy individuals and lung adenocarcinoma patients. Using the 2-D and 1-D microLC-MS/MS analyses, approximately 250 and 100 different proteins were detected, respectively, in each HSA- and IgG-depleted sample, which corresponds to only 0.4 microL of blood plasma. Automatic operation enabled the completion of a single run of the entire 1-D and 2-D microLC-MS/MS analyses within 11 h. Investigation of the data extracted from the protein identification datasets of both healthy and adenocarcinoma groups revealed that several of the group-specific proteins could be candidate protein disease markers expressed in the human blood plasma. Consequently, it was demonstrated that this high-throughput microLC-MS/MS protein profiling system would be practically applicable to the discovery of protein disease markers, which is the primary objective in clinical plasma proteome projects.

Adenocarcinoma↗

PubMed2004

Fully automated online multi-dimensional protein profiling system for complex mixtures.

For high throughput proteome analysis of highly complex protein mixtures, we have constructed a fully automated online system for multi-dimensional protein profiling, which utilizes a combination of two-dimensional liquid chromatography and tandem mass spectrometry (2D-LC-MS-MS), based on our well-established offline system described previously [K. Fujii, T. Nakano, T. Kawamura, F. Usui, Y. Bando, R. Wang, T. Nishimura, J. Proteome Res. 3 (2004) 712]. A two-valve switching system on a programmable auto sample injector is utilized for online two-dimensional chromatography with strong cation-exchange (SCX) and reversed-phase (RP) separations. The SCX separation is carried out during the equilibration of RP chromatography and the entire sequence of analysis was performed under fully automated conditions within 4 h, based on six SCX fractionations, and 40 min running time for the two-dimensional RP chromatography. In order to evaluate its performance in the detection and identification of proteins, digests of six standard proteins and yeast 20S proteasome have been analyzed and their results were compared to those obtained by the one-dimensional reversed-phase chromatography system (ID-LC-MS-MS). The 2D-LC-MS-MS system demonstrated that both the number of peptide fragments detected and the protein coverage had more than doubled. Furthermore, this multi-dimensional protein profiling system was also applied to the human 26S proteasome, which is one of the highly complex protein mixtures. Consequently, 723 peptide fragments were identified as 31 proteasome components, together with other coexisting proteins in the sample. The identification could be comprehensively performed with a 63% sequence coverage on an average, and additionally, with modifications at the N-terminus. These results indicated that the online 2D-LC-MS-MS system being described here is capable of analyzing highly complex protein mixtures in a high throughput manner, and that it would be applicable to dynamic proteomics.

Automation↗

PubMed2004

Comprehensive analysis system using liquid chromatography-mass spectrometry for the biosynthetic study of peptides produced by cyanobacteria.

Microcystins are hepatotoxic heptapeptides and general tumor promoters produced by several species of the genera Microcystis, Anabaena, Oscillatoria and Nostoc. They are non-ribosomally synthesized via a mixed polyketide synthase/non-ribosomal peptide synthetase system called microcystin synthetase. We have carried out the detection, isolation and structural determination of non-toxic peptides produced together with microcystins by toxic cyanobacteria, which are classified into several groups on the basis of their structures and some of these non-toxic peptides are also non-ribosomally synthesized as well as microcystins. In the present study, we tried to correlate the secondary metabolic peptides produced by the hepatotoxic cyanobacteria with the corresponding peptide synthetase genes. An analytical method using LC-electroscopy ionization MS and photodiode array detection was developed for the exhaustive screening of cyanobacterial peptides in Japanese strains and it was successfully applied to the peptide fractions extracted from these strains. The established method was advantageous over conventional ones using the usual HPLC and matrix-assisted laser desorption ionization time-of-flight MS, because more structural information could be obtained and it is easier to distinguish microcystins from other peptides using this method. Small amounts of other peptides could also be detected by this method. The established method will contribute to the investigation of the relationship between genes encoding the peptide synthetase and secondary metabolic peptides.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Multidimensional protein profiling technology and its application to human plasma proteome.

In clinical and diagnostic proteomics, it is essential to develop a comprehensive and robust system for proteome analysis. Although multidimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems have been recently developed as powerful tools especially for identification of protein complexes, these systems still some drawbacks in their application to clinical research that requires an analysis of a large number of human samples. Therefore, in this study, we have constructed a technically simple and high throughput protein profiling system comprising a two-dimensional (2D)-LC/MS/MS system which integrates both a strong cation exchange (SCX) chromatography and a microLC/MS/MS system with micro-flowing reversed-phase chromatography. Using the microLC/MS/MS system as the second dimensional chromatography, SCX separation has been optimized as an off-line first dimensional peptide fractionation. To evaluate the performance of the constructed 2D-LC/MS/MS system, the results of detection and identification of proteins were compared using digests mixtures of 6 authentic proteins with those obtained using one-dimensional microLC/MS/MS system. The number of peptide fragments detected and the coverage of protein sequence were found to be more than double through the use of our newly built 2D-LC/MS/MS system. Furthermore, this multidimensional protein profiling system has been applied to plasma proteome in order to examine its feasibility for clinical proteomics. The experimental results revealed the identification of 174 proteins from one serum sample depleted HSA and IgG which corresponds to only 1 microL of plasma, and the total analysis run time was less than half a day, indicating a fairly high possibility of practicing clinical proteomics in a high throughput manner.

Blood Proteins↗