• 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 the sample as well as a second aliquot (i.e. split a gel band) and enzymatically digest each with a different protease. 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. We use this strategy to search for terminal residues from protein cleavage products as well.

Proteins from biological samples can be identified and quantified after enzymatic digestion by chromatographic separation and tandem mass spectrometry. Simple protein identifications where a single protein is the dominant species can be done quickly with minimal instrument time. More complex samples require a longer analysis due to the larger number of proteins present. For immunoprecipitations and whole cell lysates we often employ a second dimension of chromatography where the sample undergoes chromatographic separation with fraction collection followed by conventional chromatography/ mass spectrometry of the fractions (MudPIT/ 2D-LC). This latter strategy can also be used for gels by subdividing gel lanes into multiple gel regions. The resulting mass spec data is then database searched for protein identification and can be quantified by various methods such as spectral counting, label free quantitation and isobaric tag quantitation.

• Protein Identification from gel bands and solutions
  SDS-PAGE gel bands can be excised, digested and analyzed for protein identification as well as solution based samples. This method is often employed for confirmation of known proteins, identification of spurious bands that may appear on a gel as well as a way to identify cleavage products.

• Solution based - Label Free and Isobaric Tag Quantitation
  Solution based samples can be digested directly or after a precipitation step and analyze. This most often employed for immunoprecipitated samples (IP-MS) and whole cell lysates. Depending on the depth of coverage needed we most often analyze the digested sample either by using a longer analysis time or by performing an initial HPLC separation with fraction collection where each fraction is analyzed by conventional chromatographic separation and mass spectrometry (MudPIT/ 2D-LC ). For samples labeled with isobaric tags (TMT - iTRAQ) we use the MudPIT/ 2D-LC strategy. For label free samples it can be advantageous to analyze a 20% aliquot of the digested sample using the direct method while reserving the remaining 80% for MudPIT/ 2D-LC analysis if greater depth of coverage is desired.

  The direct analysis method works best for samples not overly complex and 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 does not tolerate certain detergents, particularly SDS and high concentrations of Triton X100. Proteins in unfriendly buffers can be precipitated by trichloroacetic acid (TCA), as well as other precipitation methods and resuspended in appropriate buffers prior to enzymatic digestion.
  For sample buffers containing SDS (laemmli, CHAPS) we like to run into an SDS-PAGE gel for one centimeter, excise and digest which works well for both label free and isobaric tag strategies.

• 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 artefactual bands (IP bait, IgG, FLAG, etc) separately as these can suppress the signal from lower abundance species.

  We've also employed the gel based strategy without subdividing lanes for samples containing detergents unsuitable for solution based enzymatic digestion and this works well for samples fated for isobaric labeling.

Relative quantitation of proteins in multiple biological samples is obtained using either our Orbitrap Elite or Exploris 480 systems. We can perform Label Free quantitation where samples are analyzed separately and the area from individual peptides is used to compare protein concentrations across multiple samples.

The Label Free Quantitation is the same as that described under Comparative Proteomics and is typically performed on our Orbitrap Elite.

More rigorous methods of quantitation involve isotopically labeled amino acids (SILAC) or isobaric mass tags (TMT/ iTRAQ) 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 and Solution 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.


• 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 peptides are separated according to isoelectric point and fractions are collected. Each fraction is then analyzed by nano scale chromatography/ tandem mass spectrometry analysis. Please inquire prior to submitting samples to ensure the solution based samples are amenable to chemical labeling.


• 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.

Amino acids modifications (phosphorylation, alkylation, ubiquitination, glycosylation) occur frequently and are of interest. We can identify many of these from the data acquired under standard mass spec acquisition parameters using both the Orbitrap Elite and Exploris 480 systems. Labile modification such as phosphorylation and larger modifications such as ubiquitination and glycoslylation tend to be more difficult to detect using our standard methods but enrichment technologies have been developed as an aid to their detection.

The information we need to look for a given modification you might be interested in is the protein sequence or accession number, amino acid that may be modified and the additional molecular weight of the modified amino acid (add weight) which we can usually provide. With this information set the software such that the amino acid of interest can have its native molecular weight or the modified molecular weight.

• Global Searches for PTMs post acquisition
  We can revisit samples that have been previously analyzed PTMs. This works best for smaller non-labile amino acids as well as phosphorylation. The ability to detect individual peptides, particularly those that are at low abundance, is impacted by the number of other proteins present in the particular sample as low abundance peptides signal can be suppressed in the presence of high abundance peptides.

• PTM Detection on Purified Proteins
  Detecting PTMs from purified protein preparations provides more confident identifications given the signal suppression issue mentioned above as the number of post digestion peptides is much smaller. We can readily detect many PTMs with molecular weights below 200 Da. As the PTM add weight increases above this the software struggles to provide positive identifications.

• Enrichment Strategies for Global PTM analysis
  Assay kits for phosphorylation, ubiquitination/ sumolation and other modifications are available. These kits take advantage of a physical or biological characteristic of the PTM side chain to catch the modified amino acids followed by wash and elution steps to enrich the modified peptides which are then subject to tandem mass spec.

  Typically we have not been providing global enrichment services for complex samples such as IPs and whole cell lysates due to FTE capital involved in validating the results and generating reports.
  I would be more than happy to discuss enrichment strategies further and how it relates to a project you may be contemplating.

Crosslinking proteins with different chemical linkers can provide structural information related to the three dimensional conformation of protein complexes. Crosslinked peptides present identification challenges using the standard protein identification bioinformatic tools given their non-linear structure which results in multiple N and C termini. Running the sample on the instrumentation after digestion is straightforward and doesn’t require non-standard instrument parameters. We’ve found best results from gel purified species as both provides a visual of the crosslinking efficiency as well as minimizing contaminant proteins and inefficient and partial formation crosslinked species which can complicate the data analysis.

Specialized software has been developed to meet these bioinformatic challenges and we have some modest experience with several software suits. We typically use multiple results from different software suites to increase confidence. Our current favorite is Protein Prospector (UCSF) and we have several others.

We’d be happy to discuss projects you may have in mind and would welcome the opportunity to evaluate our software tools on real samples and generally increase our expertise in this field. Please contact Eric regarding sample submission or to discuss further.