Solution-based LC MS/MS technology for Protein biomarker Identification We specialize in the identification of proteins, differentially- expressed in normal vs. disease-specific biological samples.
We have developed serum and tissue-based methods for the solution-based isolation and identification of protein biomarkers, using chromatographic techniques linked to mass spectrometry.
We can provide your laboratory with the capacity to perform differential identification of proteins within sample sets, and the generation of candidate biomarkers.
Proteins are identified using our proprietary solution-based methodology (LC MS/MS). Protein mixtures are first proteolytically-digested into small peptides of 6 to 20 amino acids in length. Then, the resulting peptide mixture is separated by nanoflow reversed phase (RP) chromatography, the eluting peptides directly ionized via microelectrospray ionization, and then analyzed by tandem mass spectrometry (MS/MS) using a ThermoFinnigan LCQ DECA ion trap.
Mass spectra acquired in this manner are used to search databases and to identify known proteins. Once the proteins are identified from the disease-specific samples, a comparison of the proteins identified is performed, resulting in the identification of differentially- expressed candidate protein biomarkers. Peptide sequence analysis is performed and the spectra searched against protein databases for identification. Upon identification, these proteins are then validated by manual confirmation of MS/MS spectra, comparison of peptide fragmentation patterns and/or co-elution experiments with synthetics standards. These extra steps of validation assure a more accurate identification of the protein.
Where appropriate, we include other techniques, including, 2 dimensional gel electrophoresis that provides a map of all the serum proteins, multidimensional protein identification technology (MudPIT), which utilizes 2 dimensions of chromatographic separation, strong cation exchange (SCX) and reverse phase (RP), before the peptides enter the mass spectrometer resulting in a reduction of the complexity of the sample.
- Identifying differential protein expression between sets of Tissue, sera or other biologically-complex mixtures.
- Bands containing multiple proteins derived from SDS-PAGE gel
- Large protein breakdown products derived from 2DE or SDS-PAGE gels Small acidic proteins derived from 2DE or SDS-PAGE Analysis of cellular fractions (organelles, membrane isolations, etc) Immunoprecipitation fractions
- LC/MS/MS is also recommended for use in biological samples where proteins were not identified by MALDI/MS, often because too few peptides are observed by MALDI to give a confident ID.
- Our database searching programs such as Sequest can be used to identify posttranslational modifications, such as phosphorylation, methylation, etc.
- Organism- specific protein and nucleotide databases can be generated to focus the search and to reduce the search time.
- Identification of phosphopeptides is achievable with on-line IMAC