• Simple Protein ID from Gel Bands or Solution Based Samples
    We can identify the protein(s) present in a gel band or solution using our Thermo Fisher Orbitrap Elite mass spectrometer. Samples are enzymatically digested, the resulting peptides are chromatographicaly separated and introduced into the Orbitrap where peptides are mass measured within 3 ppm and subsequently fragmented in a low resolution ion trap. The resulting spectra are searched against a database and peptide/ protein identifications are determined.

  • In-Depth Protein Identification
    Often times a specific residue or cleavage site in a protein needs to be determined. We can digest a second aliquot (i.e. split a gel band) and enzymaticaly digest with different proteases. The goal here is to increase coverage of the entire protein through overlapping peptides. This is helpful when the residue of interest occurs in a tryptic peptide which may be less than ideal for identification by LC/MS due to size, hydrophobicity or incomplete fragmentation.

Proteins from complex biological samples can be compared after enzymatic digestion by chromatographic separation and tandem mass spectrometry. Biological samples can be separated by SDS-PAGE and the gel lanes subdivided into smaller regions/ bands. These are then enzymatically digested and analyzed by nanoscale chromatography and tandem mass spectrometry. Alternatively, we can perform a solution digest on soluble sample preps and analyze this on a long or intermediate instrument method. The resulting data is then database searched and the number of peptides from each identified protein is presented in a spreadsheet format where semi-quantitative information from the many proteins across multiple samples can be compared by the Spectral Counting Method. We can also use this same database search results in a quantitive fashion for Label Free Quantition.
We've also employed the gel based strategy withuot fractionation where the sample is run 1 to 2 cm into the gel and excised. This works well for samples containing detergents/ additives which preclude a solution based enzymatic digestion.

  • Gel based - Spectral Counting and Label Free Quantitation
    We can analyze SDS-PAGE based samples where each lane is subdivided into 4 to 10 or more molecular weight regions with care taken to excise artifactual bands (IP bait, IgG, FLAG, etc) separately. These are enzymatically digested and analyzed by nano scale chromatography/ tandem mass spectrometry and the resulting data is searched against a database for protein identification. Various methods of quantitation can be used for the resulting protein identifications including both Spectral Counting where the total number of peptides from a protein is used for quantitation and Label Free Quantitation where quantitation is based on the areas or intensities from individual peptides. Both Spectral Counting and Label Free Quantitation results can be viewed in the same Scaffold Results file.

    We've also employed the gel based strategy without subdividing lanes for samples conatining detergenents unsuitable for solution based enzymatic digestions.

  • Solution based - Spectral Counting and Label Free Quantitation
    The same analytical strategy used for gels is also applicable to solution based samples but without the fractionation step. This method works best for samples which do not require fractionation as well as samples that do not have high stoichiometric amounts of immunoprecipitation protein artifacts (IgG, FLAG, IP bait proteins, etc.) present as these tend to suppress the signal of lower abundant proteins. The digestion protocol for solution based samples is less forgiving than the gel based analysis and does not tolerate certain detergents, particularly SDS and Triton X100. Trichloroacetic acid (TCA), Acetone or chloroform/methanol extractions may be employed prior to digestion. The solution based strategy can also miss lower abundant proteins in the presence of high abundant proteins but often suffices for relatively simple protein mixtures.

    We do have the capability to do an HPLC based fractionation step of post-digestion peptides using basic RP-HPLC with fraction collection

Relative quantitation of proteins in multiple biological samples is obtained using our high resolution Orbitrap Elite. We can perform Label Free quantitiaton where samples are analyzed separately and the area from individual peptides is used to compare protein concentrations across multiple samples. The Label Free Quantition is the same as that described under Comparative Porteomics.

More rigorous methods of quantitation involve isotopicaly labeled amino acids or isobaric mass tags where samples are labeled, combined and analyzed in parallel. These methods have the advantage of multiple biological samples being analyzed simultaneously resulting in more accurate quantitation due to less experimental bias.

  • Orbitrap Elite (Gel Based - Label Free Quantitation)
    We analyze gel based samples using our high resolution Thermo Fisher Orbitrap Elite. Quantitation is based on the Label Free Quantitation method where peak areas form individual peptides are calculated and used for comparisons of relative protein concentrations between different biological samples. The work-flow is identical to the gel based Comparative Proteomics approach.

  • Orbitrap Elite (Solution Based - Label Free Quantitation)
    As an alternative to SDS-PAGE separation of proteins prior to mass spectrometry, solution based samples can be digested and the proteins are analyzed by nano scale chromatography/ tandem mass spectrometryanalysis.

  • SILAC (Stable isotope labeling by amino acids in cell culture)
    Two or more cell cultures can be grown under identical conditions except that one contains unlabeled amino acids and the others contain labeled (13C and 15N) amino acids. As the cells grow, the SILAC amino acids are incorporated into newly synthesized proteins. The samples are then combined such that both samples undergo identical lysis, immunoprecipitation, digestion, chromatography and tandem mass spectrometry. Proteins from the two samples can then be differentiated and relative quantitative information obtained. SILAC represents the current best and most accurate quantitative mass spectrometric methodology. This method is particularly well suited to gel based proteomics but can also be performed on solution based samples.

  • Quantitative Mass Tags (iTRAQ, TMT)
    Peptides can be labeled after enzymatic digestion with isobaric tags. These have the same mass but produce a low molecular weight reporter ion during tandem mass spectrometry. After enzymatic digestion, different biological samples are labeled with the isobaric reagents and mixed together. These are then analyzed and the relative abundance of the reporter ions is used for relative quantitation. Reagents for this include iTRAQ (isobaric tags for relative and absolute quantitation) and TMT (tandem mass tags). This analysis often requires an additional chromatographic step where peptideds are separated according to isoelectric point and fractions are collected. Each fraction is then analyzed by nano scale chromatography/ tandem mass spectrometryanalysis. Please inquire prior to submitting samples to ensure the solution based samples are amenable to chemical labeling.

Once transcribed, the amino acids many proteins are modified (phosphorylation, alkylation, ubiquitination, glycosylation). With our high resolution Orbitrap Elite we can identify many of these. The technology is better suited for modifications which are lower in molecular weight and becomes somewhat limited with regard to multiple heterogeneous modifications on the same peptide.

  • Post Acquisition Bioinformatics
    Many proteins have post-translational modifications. We can search for many modifications after data acquisition by re-searching the data. To perform this search we need to know the add weight (molecular weight added to the modified amino acid) and which amino acids are modified. This works better for lower molecular weight modifications and a limited number of modifications. Sequence coverage of the modified protein needs to be extensive and it is often beneficial to preform multiple sample digestions.

  • Immobilized Metal Affinity Chromatography (IMAC) for the Enrichment for Phosphopeptides
    Phosphorylated peptides tend to be difficult to detect due to low stoichiometry and poor ionization. We can enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC) prior to tandem mass spectrometry to increase the detectability of phosphopeptides.

We'd be happy to discuss non-standard projects where we may be able to utilize our low resolution Thermo LTQ mass spectrometer in conjunction with our Dionex nanoflow or Shimadzu conventional flow HPLC systems. Please inquire well in advance about these services.

For metabolite profiling services visit the Metabolite Profiling Core Facility