Mass spectrometry supports protein and peptide identification which facilitates no more tedious work for time consuming gel preparation. Creative Proteomics has been long focused on optimizing our mass spectrometry technology platform. We have developed a comprehensive protein identification pipeline, which processed with sample preparation, protein separation and purification, followed by mass analysis, and finally to systematic bioinformatics analysis.
The following picture illustrates a typical workflow for protein identification and characterization using MS/MS method. The starting point is a given protein sample that may be a single protein or a complex mixture of different proteins. An enzyme, often trypsin, is used to digest the proteins to peptides. In most cases, one or more stages of chromatography are applied to regulate the flow of peptides into the mass spectrometer. Peptides are then selected for one at a time in the first stage of mass analysis. Each isolated peptide is induced to fragment by collision in the second stage of mass analysis. The fragments are captured to synthesize an MS/MS spectrum.
With years' experience in advanced experiment equipment, Creative Proteomics can provide a variety of proteomics services to assist your scientific research, including:
As one of the leading omics analysis service provider, Creative Proteomics provides a series of protein identification services for our customers. Our service guarantees accurate and reliable results, at quick turnaround time! Please feel free to contact us and see how we can help to address your problems.
What type of samples can be analyzed?
Any sample that contains proteins and peptides can be analyzed, however, the method used will depend on the type of sample and results required.
Creative Proteomics provides analysis of proteins and peptides and their posttranslational modifications.
How should the sample be prepared for MS analysis?
The protein must be either a gel band or lyophilised. Proteins on PVDF membrane cannot be analyzed by mass spectrometry. The spot or band should be stained (e.g. with Coomassie or MS compatible silver stain) and then cut out. The gel piece should be air dried in a micro-centrifuge tube. The protein is then relatively stable and can be sent by post/courier. Providing the sample is pure and in a low salt buffer e.g. 50mM ammonium hydrogen carbonate; for TRIS or phosphate buffers the maximum salt strength is 20mM. NaCl and surfactants (e.g. SDS) cannot be present. The sample must be freeze dried or lyophilised in a micro-centrifuge tube prior to shipping.
NOTE: The type of buffer and the volume of the liquid before drying must be stated on the Sample Form. Care must be taken to avoid contamination of the gel with contaminants.
How does protein identification by mass spectrometry work?
The protein is digested into peptides with trypsin, then each peptide is ionised and analysed individually in the mass spectrometer. This process fragments each peptide by breaking off amino acids and the resulting fragmentation spectra is compared to databases using the Mascot search algorithm to look for identical amino acid sequences.
This process can identify a known protein, or closely related proteins.
NOTE: For samples which cannot be identi!ed by automatic database analysis refer to de novo sequencing.
Protein identification by mass spectrometry.
Mass spectrometry is the state of the art technique for obtaining protein sequence information because it is fast and cost effective.
To identify 1 protein use: Protein identification by mass spectrometry using MALDI-TOF/TOF (PMF+MS/MS).
To identify 1 or 2 low abundance proteins use: Protein identification by mass spectrometry using electrospray LC/MS/MS.
To identify 10s (to >100) of proteins use: Proteome mapping (MuDPIT) 1D LC-MALDI .
To identify 100s (to >1000) of proteins use: Proteome mapping (MuDPIT) 2D LC-MALDI .
To compare two (or more) samples containing 100s (to >1000) of proteins use: Differential expression by iTRAQ analysis (this provides both identities and quantification of proteins simultaneously).
Should I choose MALDI TOF/TOF or LC-MS/MS?
If the band is Coomassie stained use: Protein identification by mass spectrometry (sequencing by MS/MS) and select MALDI-TOF/TOF (PMF+MS/MS) mass spectrometry with automatic database analysis. This is the fastest and most cost effective method, and optimal for Coomassie stained gels and 2D gel spots.
If the band is silver stained or might contain more than one protein use: Protein identification by mass spectrometry (sequencing by MS/MS) and select electrospray (LC/MS/MS) mass spectrometry with automatic database analysis. This is the most sensitive method.
Is it important to know which genome database to search?
Protein identification is achieved by matching the peptide fragmentation pattern to sequences in the public databases. This is ideal for human, mouse, rice, arabidopsis, fruit, etc. If the sample is from organisms highly similar to known genomes then good results are achieved.
* For Research Use Only. Not for use in diagnostic procedures.