Services
In iLab, choose ‘General request for LC-MS/MS analysis’ for the following services:
Protein identification, in-gel
A highly purified protein, visible by Coomassie, is submitted in-gel. Proteins are digested with trypsin then extracted peptides are analyzed by LC-MS/MS. Data is searched against a publicly available and/or customer provided protein database using Sequest within Proteome Discoverer software.
Post-translational modification (PTM) site mapping
Identification/localization of PTMs on purified proteins, which can be submitted either in-gel or in-solution. Examples of PTMs include: Phosphorylation, ubiquitination, acetylation, methylation and glycosylation. If you’re interested in a specific modification, please contact us so that we may direct you to sample preparation protocols that could be used to optimize its detection.
Proteins are typically digested with trypsin (in-gel or in-solution) then analyzed by LC-MS/MS. Additional proteases can be used to maximize chances of detection, depending on protein sequence. For phosphorylation, samples may be enriched for phosphorylated peptides prior to LC-MS/MS. Data is searched against a publicly available or customer provided protein database using Proteome Discoverer.
To confirm PTMs on specific residues, please contact the MAP Core staff. We may be able to use targeted methods to specifically look for the peptide containing the PTM of interest.
Protein characterization
This service can include the following types of analyses:
1. Identification of other types of covalent modifications besides PTMs, such as a drug or other compound. The customer must provide us with the compound formula of these modifications.
2. Identification/confirmation of a mutated amino acid, dependent on location of mutated amino acid.
3. Identification/confirmation of truncated protein, dependent on protein sequence.
Proteins are typically digested with trypsin (in-gel or in-solution) then analyzed by LC-MS/MS, but other proteases can be used. To determine the detectability of a particular site on the protein, we ask you to provide the protein sequence before dropping samples so we can perform an in silico digestion.
Data is searched against a publicly available or customer provided protein database using Byonic and Proteome Discoverer.
Proteomic profiling
Identification and quantification of proteins in a complex mixture, which can be submitted either in-gel or in-solution. Proteins are digested with trypsin, then analyzed by LC-MS/MS using the QExactive HF, Exploris480, or Lumos platform. Typically, a longer gradient is used to identify as many proteins as possible. Afterward, data are searched against a publicly available protein database using either Mascot, Sequest, or Andromeda search engines. Data is processed in Proteome Discoverer, MaxQuant, or Spectronaut (DIA-based quantitation). Depending on sample complexity, hundreds to thousands of proteins can be detected in a single analysis. Quantitation allows measurement of the relative changes in protein abundances between controls and samples to identify significantly regulated proteins.
This service can be coupled with TMT-based quantitation and high pH reversed phase peptide fractionation to maximize the number of peptide/protein identifications. Up to about 9,000 proteins can be quantified with this approach. Please contact the staff for details.
Post-translational modification (PTM) profiling
Our most common PTM profiling service is global quantitative phosphoproteomics. A typical global phosphoproteomics workflow involves TMT-based quantitation, offline HPLC fractionation, and High Select FeNTA enrichment. Phosphopeptides are analyzed using the Exploris480 platform and data are processed in Proteome Discoverer. Depending on sample complexity, hundreds to thousands of phosphopeptides can be detected in a single analysis. In some cases, > 20,000 phospho sites can be detected.
Additional PTMs such as ubiquitylation, acetylation, and methylation (using Cell Signaling Technology’s antibody-based kits) can also be profiled on a global level. Please contact Laura Herring and Allie Mills regarding these services.
Affinity purification-mass spectrometry (AP-MS) analysis
Identification of protein binding partners of proteins captured using affinity purification techniques (including endogenous, overexpressed, proximity labeled, DNA, RNA, drug pulldowns, etc). There are critical sample preparation steps that must be considered before submitting these types of samples, so please contact the staff to discuss the project prior to sample preparation. Each sample MUST be submitted with a proper control.
Depending on the pull-down protocol, affinity purified proteins are digested using an in-gel, in-solution, or on-bead digestion protocol; then analyzed by LC-MS/MS. The gradient length may be extended depending on sample complexity. Afterward, data are searched against a publicly available protein database and further analyzed using MaxQuant or Proteome Discoverer. Data reports include a list of all proteins identified / quantified, as well as statistical analysis identifying interactors.
Please contact Allie Mills for further information regarding AP-MS experimental design.
Multiplex inhibitor bead coupled to mass spectrometry (MIB-MS) kinome profiling
This service is performed in collaboration with Dr. Lee Graves’ lab, please contact him with inquiries. Immobilized pan kinase inhibitors are used to capture kinases from a complex sample. Over 200 kinases can be enriched, which are subsequently digested with trypsin and analyzed by LC-MS/MS using the QExactive HF platform. Afterward, kinases are identified and quantified using MaxQuant. Relative quantitation of kinases is performed to identify kinome changes between samples. Biological replicates are required.
Targeted analysis
Analysis by LC-MS/MS using targeted methods to confirm and/or quantify a specific analyte of interest such as the detection of peptides of interest in a complex sample. Parallel reaction monitoing (PRM) mode on the QExactive HF is used to select peptides for HCD fragmentation. Data is processed using Skyline. If applicable, synthetic peptides can be used for detection and/or absolute quantitation and must be provided by the customer. Please contact the staff to discuss your project to determine the best experimental design for your project goals.
*Quantitation*
The analyses described above are quantitative. Quantitation of peptides/proteins is typically performed to compare a control to a sample (e.g. post-drug treatment, post-knockdown, etc.). BIOLOGICAL REPLICATES ARE REQUIRED. Please contact the MAP Core staff to discuss experimental design. Types of quantitation include:
1. Isobaric tagging (TMT). The facility provides the labels and will perform the labeling step – up to 18 samples can be multiplexed.
2. Label-free Data Dependent Acquisition (DDA) using area under the curve or spectral counting. Each sample is analyzed individually. No additional sample preparation required.
3. Label-free Data Independent Acquisition (DIA) using peak areas in a library-based or Direct-DIA approach. Each sample is analyzed individually.
4. Absolute quantitation using heavy labeled peptides for targeted analysis of specific peptides/proteins *only for targeted analyses (see above).
Proteins are typically digested with trypsin then analyzed by LC-MS/MS. Peptide/proteins are identified and quantified using MaxQuant (label-free DDA, APMS projects only), Proteome Discoverer (TMT, label-free DDA), or Spectronaut (Label-free DIA). Further data analysis is conducted in Perseus, and/or R. The MAP Core can provide publication quality figures such as bar graphs, volcano plots, PCA plots, hierarchical clustering with heat maps, pathway enrichment plots, as well as perform bioinformatics analyses using DAVID or IPA, among other programs.