Services:

Protein Identification:

In the past decade there has been a revolution in the application of mass spectrometry for the analysis of proteins and peptides. This has been facilitated in part by two ionization techniques, which are capable of producing charged gaseous ions of both peptides and proteins. Electrospray ionization (ESI) involves spraying a liquid solution of the peptide through a fine capillary held at a high potential. This produces charged droplets that are then rapidly desolvated producing charged ions of the peptide, which are then usually directed into a quadrapole type mass analyzer. The other ionization technique, matrix-assisted laser desorption ionization (MALDI) involves co-crystallizing the sample with an organic acid which strongly absorbs UV laser light. Upon irradiation under vacuum there is an energy transfer from matrix to peptide analyte, which produces gaseous ions that are typically measured by a time-of-flight (TOF) mass analyzer.
To understand many cellular processes it is necessary to rapidly identify components of an organism's proteome (expressed gene products). The sequencing of the genomes of many model organisms are complete which means that complete protein databases are now available for these organisms. This has precipitated a revolution in the application of mass spectrometric techniques for the identification of proteins in the databases (proteomic mass spectrometry). The identification approach involves digesting as little as 1 femptomole of gel purified protein with a site-specific protease (i.e. trypsin) and then mass analyzing the resultant peptides. The type of mass analysis can be either MS and or MS/MS. MS analysis involves the collective mass analysis of several peptides (i.e. Peptide Mass Fingerprint). MS/MS analysis is a measurement based on a single peptide and the fragments derived from it via a collisional or decay process. Once the precise mass measurements have been acquired (as in the case of the MALDI-QIT-TOF better than 10ppm mass accuracies) they are compared against the various databases using licensed search algorithm software. The program that the laboratory uses for this analysis is Mascot ( www.matrixscience.com ). This search algorithm generates theoretical mass profiles for each protein/peptide in a specified database and compares it with the experimental data to arrive at a statistical correlation. MS types of identifications are successful with relatively pure protein samples where several peptides point to a single protein component. When there are several proteins or very few peptides generated as in the case of low abundance samples then MS/MS analysis is advantageous since it is based on sequence related information from a single peptide which can be used to map back to one of the original protein components. In addition MS/MS data can be used to search nucleic acid databases like the Expressed Sequence Tag (EST) databases or Genomic databases.
The laboratory is equipped with both a Shimadzu Biotech Axima TOF²and Axima QIT MALDI-TOF instrumentation, which are currently being used to identify proteins by both MS data from peptide mixtures as well as MS/MS data derived from Collisional-Induced-Dissociation (CID) analysis of individual peptides which have been selectively gated from the mixture. The laboratory also has a Thermo LCQ Deca ESI Ion Trap (LC/MS/MS) mass spectrometer obtained with generous support from the Howard Hughes Medical Institute. The instrument is directly coupled to a capillary HPLC system. The MALDI -TOF systems are used for most gel-purified samples since it has higher throughput and sensitivity. The ESI ion trap is used for samples in larger abundance which shows greater complexity due to multiple protein components since peptides are prefractionated prior to having there MS/MS spectras recorded

Sample preparation for protein identification:

Samples can be prepared as either 1D gel bands or 2D gel spots. If you need to use a 2 D gel service I recommend either Kendrick Labs or the Protein Fractionation Group located here at the University of Massachusetts Medical School. Depending on the amount of material they can be stained with standard Coomassie Blue. Alternatively samples with much lower abundance can be stained with Sypro Ruby (a fluorescent stain from Molecular Probes) or Silver. Silver staining must omit the use of gluteraldehyde in the fixing process. Most modern kits (Sigma ProteoSilver or BioRad Silver Stain Plus) are compatible with mass spectrometry. It is extremely important to remember that when preparing low abundance proteins that precautions must be taken to prevent keratin contamination. This means wearing gloves during the entire preparation process and using a clean apparatus for running gels etc. Excise bands of interest in a dust free environment and cut as closely to the staining boundary as possible because the resolution of your gel is only as good as how you cut. I also recommend that you send me a gel image so that I can asses the sample purity and abundance.