itraq Reagent Quantification using 4700 Proteomics Analyzer and GPS Explorer v2.0 Software

Transcription

1 itraq Reagent Quantification using 4700 Proteomics Analyzer and GPS Explorer v2.0 Software 4700 Proteomics Analyzer Introduction The characterization of protein mixtures continues to be a major goal of proteomics and biomarker research. This is an important factor in helping to understand complex biological systems, determine relationships between proteins such as protein function and protein-protein interactions and discover markers indicative of disease, toxicity or drug efficacy. Often the goal toward this understanding is to monitor changes in protein mixture composition under different physiologically relevant conditions, a type of study referred to as differential profiling. While monitoring these qualitative changes is valuable, there is a need to develop quantitative tools that can provide more insight into these experiments. These requirements for broad proteome coverage, multiplexing, absolute quantitation, and more statistically significant information have led to the development of the itraq Reagents. Applied Biosystems itraq Reagents are a multiplexed set of four isobaric reagents which are amine specific and yield labeled peptides which are identical in mass and hence also identical in single MS mode, but which produce strong, diagnostic, low-mass MS/MS signature ions allowing for quantitation of up to four different samples simultaneously. In addition to multiplexing, information such as post-translational modifications is not lost using this new chemistry. Since all peptides are tagged, proteome coverage is expanded and analysis of multiple peptides per protein improves the confidence in those identified. The 4700 Proteomics Analyzer and GPS Explorer v2.0 support the identification and quantification of proteins using these novel itraq Reagents. The process for generating the quantification information from the completed MS/MS analysis and joining that with the protein identification information is described herein. Key Features Support for multiplexed quantification using novel isobaric tags The rich LC MALDI feature set of the 4700 software allows for peptide separation to be applied to the complex mixtures Definitive protein ID through the high quality MALDI MS/MS data coupled with the enhanced fragmentation pattern observed for itraq Reagent labeled peptides High quality low mass data from the 4700 Proteomics Analyzer enabling accurate and precise quantification for the reporter region of the itraq Reagent tags

2 This technical note details the use of the 4700 Explorer Software and GPS Explorer Software for generating a list of definitive identifications coupled with the precise quantification from the itraq Reagent tags. The overall steps in the process include: 1)Using the 4700 to acquire MS and MS/MS data from the itraq Reagent labeled samples 2)Using GPS Explorer Software to perform a database search of the resultant MS/MS data 3)Generation of the peak areas for each signature ion and transferring them to a combined excel spreadsheet. 4)Copying the protein identification information from GPS MS/MS Summary Table over to the Excel spreadsheet. 5)Calculation of the itraq Reagent ratios Obtaining the itraq Reagent signature peak area from 4700 Using the 4700 Explorer Software, open the desired spot set and select the MS/MS run of interest. Sort MS/MS list by precursor mass by double clicking on the Precursor Mass column. Once the list is sorted in order of mass, click on the the Analysis Tab

3 Select Mass for peak selection criteria and enter 114.1, with a Tolerance +/-.2 m/z. The plot option peak attribute must be area and not cluster area. Click the Plot button and wait. The program reads every line in the Spot Set and sends the information to Excel. Note: You may get a message stating that the mass cannot be found in one or more wells. If so, click OK to continue. Upon completion of the data extraction process, Excel will open on its own.

4 The peak area column will have a generic name. Change this to reflect the reporter ion mass that was extracted to avoid confusion when the next mass is extracted and the sheets are combined for analysis. Repeat this process with each reporter ion mass (115.1, 116.1, 117.1) used in the experiment. The peak areas should be sent to the same file but different sheets. If a new incidence of Excel is opened this is OK. Cut and paste to get all 4 columns of peak area and at least 1 copy of Spot position to a single sheet in the file. Align side by side. You will be adding the data from the GPS MS/MS Summary Data file to this spreadsheet next. Check that you have the same number of rows for each column. Save this spreadsheet. Configuring GPS Explorer Software to Search using the itraq Reagent Tags The Mascot search engine used by GPS Explorer must be configured with the new itraq Reagent tags. This can be done by going to the computer running Mascot, clicking on the Start Programs Mascot Config Mascot Modification File. The Mascot modifications file will open. Edit this file to add the N-terminal and lysine modifications shown below. When using the MMTS cysteine modifier supplied with the kit, this modification needs to be added to the modifications file as well. The information can be added to the end of the file. Save the new modification file (back-up your old modification file before creating this new file). The GPS server must be restarted for the changes to take effect. Title:(N-term)_iTRAQ Nterm: * Title:Lysine(K)_iTRAQ Residues:K * Title:MMTS (C) Residues:C *

5 Using GPS Explorer v2.0 to obtain protein identifications by database searching Set-up sample set and analysis for a LC-MALDI experiment. For fixed modifications choose the Lysine(K)_ itraq, (N-term)_ itraq and MMTS (C) modifications.

6 Select MS/MS peaks staring from 150 Da on the Peak Filtering Tab. MS range should be set from m/z 900 or 1000 since itraq Reagents add 145 Da to the peptide. Click Submit button and wait. When the search has finished, click on the MS/MS Summary tab. Sort table by increasing observed precursor mass. Check that you have the relevant columns in your summary table, such as: observed mass, ion score, protein name and species name. Use right mouse button to Select All and copy all of MS/MS summary list. Open Notepad and paste in the MS/MS summary list copied from Excel, and save as text file. Open text file in Excel and follow the steps

7 Click finish. Copy the contents of this file with the contents of the area summary sheet. There is a lot of information within this file. The next step is to determine the reporter region ratios. Insert three columns and label them Corrected 114/117 ratio, Corrected 115/117 ratio and Corrected 116/117 ratio. Apply correction factor formulae in Excel. The correction factor numbers can be found in the Certificate of Analysis that comes with your reagent kit.

8 Reagent % of -2 % of -1 % of +1 % of +2 itraq Reagent itraq Reagent itraq Reagent itraq Reagent Corrected 114/117 ratio =ROUND(((A *A 115 )/(A * A 116 )),3) Corrected 115/117 ratio =ROUND(((A *A *A 114 )/(A * A 116 )),3) Corrected 116/117 ratio =ROUND(((A *A *A 115 )/(A * A 116 )),3) For Example Cell G2 =ROUND(((B2-0.02*D2)/(F2-0.06*E2)),3) Fill in the formulae for all peaks. You can now use the sort tool in Excel to sort the results by fields of interest.

9 Corrected itraq Reagent ratios Protein identification Ion score Applera Corporation is committed to providing the world s leading technology and information for life Scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. AB (Design), iscience, iscience (Design), TOF/TOF and Applera are trademarks of, and Applied Biosystems is a registered Trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries. For Research Use Only. Not for use in diagnostic procedures Applied Biosystems. All rights reserved.