It is a technique that combines high-pressure liquid chromatography with tandem mass spectrometry to identify complex mixtures of proteins and peptides. It is used to identify proteins that are potential biomarkers, for drug development research, and in forensics to identify unknown compounds. Mass spectrometers can also be used for quality control and quality assessment, and to map structure of compounds.
How are the LC-MS/MS instruments used at ITSI-Biosciences?
Once a protein is identified ITSI will also help to figure out their role in biological systems. The LC-MS/MS instruments are complementary. The LC-MS/MS instrument is more sensitive, has a faster scan speed and can analyze many samples per day. The LC-MS/MS is primarily used for identification and quantitation of low abundant proteins, and sites of post-translational modifications can also be assigned to specific amino acid residues within a protein’s sequence using the LC-MS/MS.
What kind of samples should be submitted to ITSI for protein identification?
a) Identification and annotation,
c) Post-translational modification mapping,
d) Verification of purity,
e) Detection of impurities in samples,
Both one-dimensional or two-dimensional gel bands stained with Coomassie, Silver, or Sypro stains can be submitted. The bands will be reduced and alkylated, digested with trypsin, and analyzed by LC-MS/MS. The MS/MS spectra generated during the analysis will be searched against theoretical spectra from protein databases such as the NCBI non-redundant database, the SwissProt database, and a number of databases from the International Protein Index using the SEQUEST algorithm. ITSI can also perform 1D gels, or comparative proteomics experiments using the 2D-DIGE technology, identify differentially expressed proteins, pick and digest candidate proteins with robots and identify the proteins.
Does ITSI have the capability to quantify proteins by mass spectrometry?
Yes. ITSI recently acquired a new mass spectrometer that is enabled for iTRAQ (Isobaric Tags for Relative and Absolute quantitation of proteins) the current benchmark for non-gel quantitative proteomics. With iTRAQ ITSI can simultaneously detect and quantify proteins. Either 4-plex or 8-plex experiments can be designed to allow for protein quantitation of up to 8 different sample types in a single analysis.
What is the smallest amount of protein that ITSI can detect?
At ITSI-Biosciences our systems are optimized to identify proteins at the attomole level. Any protein band that is visible by Coomassie stain will be identified in our laboratory.
How am I confident that the identification is correct?
A very stringent scoring system is used to identify peptides and proteins using the SEQUEST algorithm. Additionally, experienced mass spectrometrist’s with more than 20 years combined experience manually review the data before accepting the results.
What is the typical sequence coverage obtained?
The typical sequence coverage for “good and confident” protein identification is 30% of the protein’s entire sequence. Sequence coverage above 50% is considered great. At ITSI-Biosciences we routinely obtained sequence coverage as high as 85% for some proteins.
What is the typical turnaround time?
Turnaround time depends on what is required and the number of samples. Typically turnaround time for protein identification by LC/MS/MS is 48 hours for 1-10 samples. Turnaround time for Post Translational Modification mapping for 1-10 samples is about 5 days.
What kind of data do I get if I submit a protein sample?
You will receive a protein identification report that contains the a) sample ID, b) sample type, taxonomy, the names of the identified proteins, the accession numbers, the molecular weight and isoelectric point of the protein, the number of peptides matched to the protein, and the percent sequence coverage obtained for the protein. We also offer GO annotation including Molecular Function, Cellular Component and Biological Process of the identified protein.
Do I get a discount if I submit multiple samples?
Yes. A discount of 10 – 15% will be extended if more than 10 samples are submitted at a time. Please contact the mass spectrometry unit for more information/pricing.
How much is the cost of identifying a protein in a gel band?
The cost for protein identification depends on the sample complexity. Repeat customers and customers that submit multiple samples generally receive discounts.