- Protein Identification
- Protein Quantification
- Protein Post-translational Modification
- Protein Differential Expression
- Policies and Prices
To identify proteins in complex or simple mixtures (e.g. gel cutouts), we apply tandem mass spectrometry (LC-MS/MS) and an in-house platform IdentiXL to identify proteins on a large scale, in several formats, and establish "qualified" peptides in those samples. The proteomic sample data are acquired using a ThermoElectron LTQ, searched with SEQUEST (Bioworks), validated with TPP, compared and reported to provide useful information for the researcher.
- Label-free Quantification
An in-house software package, IdentiQuantXLTM, has been developed to provide large-scale protein identification and label-free quantification using either low or high resolution LC-MS/MS data. It is a comprehensive tool that overcomes the limitations of multi-dimensional electrophoretic approaches and stable-isotope labeling techniques. It is capable of profiling >8,500 proteins in a complex sample with a linear dynamic range of 128 fold and sub-femtomole sensitivity.
- This platform includes protein identification, validation, peptide filtering, intensity extraction, quantification, and statistical analysis of differential protein expression. In addition to including other readily available software, this package provides two new software elements, IdentiXLTM and QuantXLTM.
IdentiXLTM provides protein identification on a large scale, in several formats, and determination of qualified peptides, including their retention time and mass-to-charge ratio (m/z) for intensity extraction. QuantXLTM provides protein quantification on a large scale in several formats and includes statistical analysis. The entire package is an MS Windows-based integral workflow that overcomes the bottleneck in large scale data analysis of protein identification and label-free quantification using LC-MS/MS.
- SRM (Selected Reaction Monitoring)
To detect the presence of specific proteins in complex mixtures without the need for antibodies, SRM greatly reduces background interference and increases sensitivity and selectivity.
Differential Protein Expression
- IdentiQuantXLTM (see Protein Quantitation above)
- 2-D PAGE
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has a unique capacity to resolve complex mixtures of proteins according to charge (pI) and mass (MW). Our platform incorporates capabilities including highly parallel 2-DE (20 gels/tank, 60 gels/run) enabling reproducible gel-based differential expression analysis (including 2-D Western blots) and LC-MS/MS for protein identification, quantitation, and characterization. This unique approach greatly reduces gel-to-gel variability and renders the 2-DE gel patterns generated in this manner both physically and electronically superimposable. In turn, this enables robust comparison of individual protein abundance and reliable analysis of differential protein expression using Nonlinear Dynamics' SameSpotsTM software.
The primary strength of 2-DE is its resolving power compared to other separation techniques. Proteins with PTMs that result in altered pI (charge modifications) are readily resolved and occupy physically unique positions in the gel matrix. In fact, as modification stoichiometrically increases, the protein carrying these modifications is located physically at a greater distance from its unmodified (or partially modified) isoforms, essentially in an enriched form for easy post hoc analysis. Given the propensity for PTM abnormalities in cell injury and disease, the ability to detect, quantify, and later characterize PTM of this type is a major strength of 2-DE.
- Western Blotting
Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the nature of the protein detected, such as its molecular weight, in some cases, isoelectric point, tertiary structure and its biological activity. It also can be used to confirm protein quantification from LC-MS/MS and 2-D PAGE.
Protein Post-translational Modification
To search for all manner of post-translational modifications (PTMs) in complex sample data, we apply InsPecT. The determination of individual modifications (e.g. phosphopeptides, glycopeptides, etc.) is also possible when combined with suitable enrichment methodologies.
Last page update on 10/02/2012
For questions and potential collaboration, please contact:
Frank A. Witzmann, Ph.D.
Office Phone : 317-278-5741
Xianyin Lai, Ph.D.
Office Phone : 317-274-3499