Navigation Links
Direct On-Membrane MS/MS Analysis using the Chemical Printer (CHIP-1000) and AXIMA-QIT


Microscale on-membrane PMF analysis can be conducted on proteins transferred to a PVDF membrane utilizing the microdispensing function of a chemical printer (Refer to AXIMA Application 14, 17). Moreover, using the AXIMA-QIT ion trap MS in conjunction with the chemical printer enables MS analysis with high mass accuracy (<0.1Da). In this application, we succeeded in conducting MS/MS analysis of proteins immobilized on PVDF transfer membranes, something which has been difficult up to now due to the irregularities of the membrane surface and electrical charge. Direct on-membrane MS/MS analysis using the chemical printer together with the AXIMA-QIT, in addition to the established methodology for onmembrane PMF analysis, is expected to be extremely effective in identification of unknown proteins.

Protein spots from E. coli separated by 2-dimensional electrophoresis were transferred to a PVDF membrane for use as the sample (Fig. 1). A chemical printer was then used to perform enzymatic digestion (trypsin digestion) on selected protein spots. Following enzymatic digestion, matrix solution was microdispensed on each print position, and onmembrane MS analysis was then conducted using the AXIMA-QIT. The obtained MS spectra are shown in Fig. 2.




Next, the AXIMA-QIT was used to conduct on-membrane MS/MS analysis of precursor ions identified from characteristic signals of protein spots verified through PMF analysis. We were able to identify the respective protein spots by conducting an MS/MS ion search based on the obtained MS/MS spectra.

Fig. 1 shows a portion of the transfer membrane stained with Direct Blue 71 after transfering E. Coli extract separated by 2-dimensional electrophoresis to the PVDF membrane. The MS spectra obtained from microscale on-membrane PMF analysis using the chemical printer are shown i n Fig. 2. Combining the chemical printer with the AXIMA-QIT ion trap mass spectrometer enables, on-membrane MS analysis with high mass accuracy (<0.1Da).

Fig. 3 shows the results obtained from on-membrane MS/MS analysis of the precursor ions identified from characteristic signals (Spot 1: m/z =1610.79, Spot 2: m/z =1708.87, Spot 3: m/z =1568.85) confirmed by PMF analysis. The results of MS/MS ion search using these MS/MS spectra provided identification of these protein spots with convincing scores (Fig. 4).



Literature cited: T. Nakanishi et al., J. Proteome Res ., 2005, 4, 743-747



'"/>

Source:


Page: All 1 2

Related biology technology :

1. Counting Colonies or Plaques Directly from Images of Agar Plates
2. High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors
3. Constructing Directional cDNA Libraries from Limited Amounts of RNA
4. Generate High-Quality, Directional Plasmid cDNA Libraries
5. Optimizing the Direct Amplification of Missing cDNA 5 Ends Using the Eppendorf Mastercycler gradient
6. Optimizing the Direct Amplification of Missing cDNA 5 Ends Using the Eppendorf Mastercycler gradient
7. TripleMaster PCR System: Direct Comparison with Competitors
8. DirectPrep 96 Miniprep System for cost-effective, high-throughput plasmid DNA purification
9. A Further Step in Understanding Apoptosis Direct Detection of PARP Cleavage
10. YOUR DATA: Direct Real-time RT-PCR on Sympathetic Neuron Lysates Silencing of Focal Adhesion Kinases with Silencer Validated siRNAs
11. YOUR DATA: Direct Real-time RT-PCR on Sympathetic Neuron Lysates Silencing of Focal Adhesion Kinases with Silencer Validated siRNAs
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:10/11/2017)... ... October 11, 2017 , ... The CRISPR-Cas9 ... enabling overexpression experiments and avoiding the use of exogenous expression plasmids. The simplicity ... for performing systematic gain-of-function studies. , This complement to loss-of-function studies, such ...
(Date:10/11/2017)... LAGUNA HILLS, Calif. , Oct. 11, 2017  SkylineDx ... London (ICR) and University of Leeds ... to risk-stratify patients with multiple myeloma (MM), in a multi-centric ... The University of Leeds is the ... UK, and ICR will perform the testing services to include ...
(Date:10/10/2017)... ... October 10, 2017 , ... ... advancing targeted antibody-drug conjugate (ADC) therapeutics, today confirmed licensing rights that give ... Liposomal Nanoparticle), a technology developed in collaboration with Children’s Hospital Los Angeles ...
(Date:10/10/2017)... ... October 10, 2017 , ... ... process optimization firm for the life sciences and healthcare industries, announces a presentation ... San Francisco. , The presentation, “Automating GxP Validation for Agile Cloud Platforms,” will ...
Breaking Biology Technology:
(Date:3/30/2017)... YORK , March 30, 2017 Trends, ... type (physiological and behavioral), by technology (fingerprint, AFIS, iris ... voice recognition, and others), by end use industry (government ... and immigration, financial and banking, and others), and by ... Europe , Asia Pacific , ...
(Date:3/28/2017)... , March 28, 2017 ... Biometrics), Hardware (Camera, Monitors, Servers, Storage Devices), Software (Video ... and Region - Global Forecast to 2022", published by ... in 2016 and is projected to reach USD 75.64 ... 2017 and 2022. The base year considered for the ...
(Date:3/24/2017)... , March 24, 2017 The Controller General of ... Mr. Abdulla Algeen have received the prestigious international IAIR Award ... Continue Reading ... ... and Deputy Controller Abdulla Algeen (small picture on the right) have received ...
Breaking Biology News(10 mins):