Navigation Links
Achieving Reliable Protein Quantitation Results Using ICAT Reagents, MALDITOF Mass Spectrometry, and Advanced Data Interpretation Tool

Overview

A challenge of quantitative analysis of complex protein mixtures using isotope coded affinity tags is to correctly identify and obtain accurate quantitative information for ICAT reagent pairs on a spectrum generated by mass spectrometry. Because MALDI-TOF mass spectrometry can easily generate peptide mass fingerprint profiles and relative quantitation, it can be used to quickly screen the ICAT reagent pairs representing the up- and down-regulated proteins from complex mixtures. The challenge lies in how to distinguish true signal from noise present in the analysis.

Accuracy of the quantitative results and dynamic range depends on peak detection, data processing, and interpretation strategies. We describe here the software implemented for ICAT reagent analysis using MALDI-TOF mass spectrometry (Figure 1). Examples of ICAT reagent data analysis using this software are demonstrated and show the experimental accuracy and dynamic range.

Key Feature

ICAT reagent experiments can be performed on a MALDI-TOF instrument to achieve reliable quantitative results.

Experimental Conditions

We performed ICAT reagent experiments on proteins such as BSA, a complex 10-protein mixture using the ICAT reagent kit for protein labeling. The purified biotinylated peptides were generated from a mixture and further fractionated by reverse-phase separation and analyzed by MALDITOF mass spectrometry. Data was submitted to a software program written in Microsoft Visual Basic for Applications available for use with mass spectrometry software. The software offers intelligent features such as intensity-based peak selection based on a userdefined mass range increment. It contains mass filters that automatically remove sodium and potassium adducts and user-defined common contaminants from ICAT reagent experiments. Data processing algorithms such as advanced baseline correction, noise reduction, and de-isotoping could be applied to data automatically prior to quantitative analysis. Because of trace amounts of D7 and D6 present in the heavy ICAT reagent, the software allows for a correction factor for these species.

Results

A key element in the software is the flexibility to identify the ICAT reagent pairs based on a theoretical peptide mass list from known protein digests, which could lead to accurate identification of ICAT reagent pairs and therefore reliable quantitative results. For example, the quantitative results from the mass spectrum of the labeled(D8) vs. non-labeled(D0) digested BSA yielded a ratio of 0.91 (vs. theoretical value of 1.0) with a standard deviation of 0.12 (Figure 2 and Table 1).

Alternatively, the VBA software automatically looks for all possible ICAT reagent pairs if the protein digests are not previously identified. False positives could be deleted by applying statistics or re-evaluating the spectrum. For MALDI-TOF analysis, this suppression is often a concern for quantitative analysis because of the complex nature of the sample. Reverse phase separation can improve the dynamic range, as is demonstrated in Figure 3. In this case, the dynamic range for a peptide of interest from a 10-protein mixture is demonstrated by the software to achieve 0.11 (vs. theoretical ratio of 10:1) and 0.03 (vs. theoretical value of 30:1) accurately.

Conclusion

Using MALDI-TOF and advanced data interpretation software, users can achieve reliable quantitative results of affinity-purified ICAT reagent-labeled protein digests. This method can be used as a potential assay to rapidly screen peptides derived from the upand down-regulated proteins that are potential bio-marker candidates in complex mixtures.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Reliable Assessment of Micronuclei for Genotoxicity Testing in Mice or Rats
2. Efficient and Reliable PCR Setup Using Eppendorf MasterMix
3. Efficient and Reliable Linear Amplification of cRNA
4. A New C-Terminal GST Vector for Protein Production in S. pombe
5. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
6. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
7. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
8. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
9. Optimized Imaging of Protein Gels Stained with Coomassie Brilliant Blue Dye
10. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
11. Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... ... 2016 , ... Parallel 6 , the leading software as a service ... Virtual Patient Encounter CONSULT module which enables both audio and video telemedicine communication ... , Using the CONSULT module, patients and physicians can schedule a face to face ...
(Date:6/24/2016)...  Regular discussions on a range of subjects including policies, ... entities said Poloz. Speaking at a lecture to ... he pointed to the country,s inflation target, which is set ... "In certain areas there needs ... economic goals, why not sit down and address strategy together?" ...
(Date:6/23/2016)... ... 23, 2016 , ... UAS LifeSciences, one of the leading ... UP4™ Probiotics, into Target stores nationwide. The company, which has been manufacturing high ... its list of well-respected retailers. This list includes such fine stores as Whole ...
(Date:6/23/2016)... ... June 23, 2016 , ... Charm Sciences, ... microbial test has received AOAC Research Institute approval 061601. , “This is another ... year,” stated Bob Salter, Vice President of Regulatory and Industrial Affairs. “The Peel ...
Breaking Biology Technology:
(Date:6/15/2016)... New York , June 15, 2016 /PRNewswire/ ... a new market report titled "Gesture Recognition Market by ... and Forecast, 2016 - 2024". According to the report, ... USD 11.60 billion in 2015 and is estimated ... reach USD 48.56 billion by 2024.  ...
(Date:6/2/2016)... -- The Department of Transport Management (DOTM) of ... US Dollar project, for the , Supply and ... and IT Infrastructure , to Decatur ... of Identity Management Solutions. Numerous renowned international vendors participated in ... was selected for the most compliant and innovative solution. ...
(Date:5/20/2016)... , May 20, 2016  VoiceIt is excited ... with VoicePass. By working together, VoiceIt ...  Because VoiceIt and VoicePass take slightly different approaches ... increases both security and usability. ... about this new partnership. "This marketing ...
Breaking Biology News(10 mins):