Proteomics has been established by combination of two-dimensional gel electrophoresis and mass spectrometry. The most significant advantage of proteomics over the conventional protein analysis is the high-throughput performance in comprehensive analysis of almost all proteins expressed in a specific tissue or cell type under a given condition. Mass spectrometry plays important roles not only in identification of proteins separated by two-dimensional gel electrophoresis but also in analysis of post-translational modifications.
Application of mass spectrometry for the life sciences, especially for the proteome analysis, has attracted considerable attention as it shows high potential for the complete of comprehensive analysis of protein expressions. Although the technology progress of the mass spectrometry is outstandingly fast, there are a few difficulties to solve in the peripheral areas. In this report, we focus on the sample preparation in the up-stream of mass spectrometry and the data analysis method at the down-stream of mass spectrometry, and introduce/discuss the current and new technologies.
With the development of proteomics, biological researchers are increasingly using mass spectrometer. Hence, it is required for the analytical instruments to analyze the many kinds of biomolecules immediately and easily. The AXIMA-TOF2 TM, a MALDI TOF-TOF system features a novel, dual ion gate capable of high resolution precursor ion selection and high energy CID cell. This instrument has a superior ability of protein identification. In addition, it can be applied to the study of different classes of biomolecules. The following three features show that AXIMA-TOF2 TM can contribute to the analysis of biomolecules. 1) The unique curved field reflectron enables the acquisition of all PSD and CID fragment ion without stepping the reflectron voltage. 2) High energy CID-MS/MS can generate and detect a large amount of valuable product ions to get structural information. 3) The high performance monoPULSE ion gate can select target ion correctly from crowded MS spectra of complex samples such as mixtures and be able to measure MS/MS spectra.
Mass spectrometry has become the main technique in proteomics study. And the strategy has been recently changed to focus more on research of target proteins from comprehensive proteins, because characterization of target proteins and quantitative study of protein biomarkers could provide important knowledge for drug discovery and development. Protein biomarkers and PTM (post-translational modification) are the main targets in target protein research called focused proteomics. However, biomarker discovery and PTM research are challenging due to difficulty in detection. A phosphate group of a compound could decrease ionization efficiency, which makes the detection of phosphorylated proteins more difficult. Most biomarkers are low abundant proteins, so detection of such compounds from highly complex samples like serum and cell lysate is not easy without sample fractionation using multi dimensional LC. The difficulty of proteomics technology using mass spectrometry results from lack of a tool to detect target proteins directly in complex samples. Here we introduce a strategy to detect a target protein directly even in a low concentration sample (e.g., PTM sample and low abundant biomarker) without additional fractionation. In this strategy unique scan modes of 4000 Q TRAP® system, precursor ion scan and MRM are key features to remove contaminant signal in chromatogram. We also show successful examples of the detection of phosphorylated peptides from digested protein kinase, the verification of low abundant protein using a new tool, "MIDASTM workflow", and the monitoring for variation of phosphorylation sites using iTRAQTM reagent.
Mass spectrometer is one of the most promising instruments for proteomics and metabolomics. It is not only applied for the protein and small molecule identification, but also for the complicated post-translational modification analysis, biomarker discovery from the quantitative analysis between the multiple samples, and so forth. Hitachi High-Technologies produced a novel liquid chromatograph mass spectrometer (NanoFrontier LD), which may identify and quantitate the proteins and peptides of interest. The improvements in the detection system brought the dynamic range and resolution over 5,000 and 10,000, respectively, enabling the high-resolution determination. Alternative collision-induced dissociation technique enhances the information abundance, and functional addition to the software advances the analysis efficiency. Furthermore, we also developed the pretreatment methodology, for enzymatic digestion and affinity purification, by applying the unique protein immobilization technique presently used in protein microarrays. These technical approaches and details were described and discussed.
The mass spectrometry (MS) has been a mainstay of proteomics and metabolomics research in these days. In a field such as biomarker research, more stringent accuracy and sensitivity on MS are demanded to identify very low abundant proteins in human plasma. It is essentially needed to deplete high abundant proteins from complex human plasma but with the least no specific binding to the target proteins which is a kind of contradictory difficulty. Two dimensional SDS polyacrylamide gel electrophoresis (2D SDS PAGE) has its limitation in pH range and molecular weight to some extent and is not able to yield high through put in sample preparation. Further more conventional nano electrospray ionization (ESI) technology which is the most appropriate to analyze such low abundant proteins needs frequent adjustment for ionization. The multi dimensional protein identification technology using two dimensional high performance liquid chromatography (HPLC) technology followed by HPLC chip MS analysis can substitute 2D SDS PAGE and conventional nano ESI ionization to realize more sensitive, more accurate , ease of use and high through put analysis for proteomics and metabolomics research. And also multi affinity removal system followed by OFFGEL Fractionator make it possible to deplete high abundant proteins from complex human plasma with the least no specific binding to very low abundant target proteins and separate the mixture of such target proteins in prior to applying them to mass spectrometry. Agilent Technologies Inc developed such total analysis system from sample preparation down to biomarker search software.
Combining patented Orbitrap technology with the LTQ linear ion trap, Thermo Scientific's LTQ Orbitrap enables faster, more sensitive and more reliable detection and identification of compounds in complex mixtures. Its outstanding mass accuracy, mass resolution and reliable high sensitivity MSn performance make it a clear alternative to existing hybrid time-of-flight systems. Furthermore, Intelligent Data Dependent (IDD) instrument control provides unmatched flexibility with parallel scanning and MS/MS detection on both detector, LTQ and Orbitrap. This hybrid system displays its greatest force on Omics applications.
Based on MALDI-TOF mass spectrometry technique, Bruker Daltonics is developing the CLINPROTTM system solution for the clinical proteomics including biomarker search and Imaging MS. For the biomarker search from body fluid like blood serum or plasma, simple sample purification using CLINPROTTM magnetic beads kit is effective. After MS measurements, ClinProToolsTM software is useful to search the biomarker from many complicated spectra. On the other hands, direct monitoring of protein distribution in a tissue using Imaging MS technique is attractive. Bruker Daltonics MALDI Molecular ImagerTM is a total solution of Imaging MS using MALDI-TOF-MS. It includes flexImagingTM software, which has wizard-like interface simplifying the operations and settings from automatic measurements to data processing/evaluation. MALDI Molecular ImagerTM is also a part of CLINPROTTM system. Proteomics analysis based on mass spectrometry also is expected for post translation modification analysis. Electron transfer dissociation (ETD) in a non-linear Paul trap has been introduced as a new fragmentation technique, which avoids internal parent ion heating. Induced by the electron transfer the intermediate peptide radical cation fragments randomly at each amino acid position of the peptide backbone, which is particularly suitable for PTM identification and analysis of large multiply charged peptides. HCTultra PTM discovery systemTM has CID- and ETD-MS/MS both, which takes easy CID-MS/MS data as proteomics analysis and ETD-MS/MS data as PTM analysis. Top-down proteomics by FT-ICR-MS is very powerful technique for identification of protein and PTM analysis. Bruker Daltonics is developing Top-down analysis system solution.
Conventionally “bottom-up” approach has been applied for normal proteome analysis using mass spectrometer. This method has several advantages such as high sensitivity and accuracy in protein identification, because in this method, protein is firstly digested into peptides, which are then analyzed by using mass spectrometer. On the other hand, recently “top-down” proteomics technique is getting popular to analyze intact protein directly. This method does not require prior protein digestion process and it is expected to have usefulness of this method also for post translational modifications. By using Fourier transform mass spectrometer (FT-ICR MS), “top-down” proteomics has a great advantage such as, more accurate measurements of molecular weight of protein by using the feature of more accurate mass and isotope resolution of higher charge state, and much more qualified information by utilizing various internal dissociation methods (SORI-CID, IRMPD, ECD or any combination). This presentation focuses on analyzing intact protein mixture to aim “top-down” proteomics. Mixture of several intact proteins is analyzed using FT-ICR MS with no pretreatment such as sodium dodecyl sulfate polyaclylamide gel electrophoresis or high performance liquid chromatography. The dissociation of intact protein inside of FT-ICR MS can generate a lots of useful information.