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Intact Mass Analysis by MALDI-TOF

Protein or peptides are submitted lyophilized or in-solution – please contact us for buffer and concentration requirements. Intact molecular weight can be determined by MALDI-TOF/TOF (low resolution), mass accuracy is dependent on the molecular weight range of the analyte. The higher the molecular weight, the lower the mass accuracy. Other molecules such as small molecules (ie, drug compounds), polymers, oligonucleotides and synthetic proteins/peptides may be submitted.

MALDI-TOF Open Access Training

Due to COVID-19, the MALDI-TOF is only accessible as a walk-up instrument for previously trained users. Please contact Laura Herring with questions.

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 either Mascot or Sequest search engines. Data is processed in Proteome Discoverer or MaxQuant.

To confirm PTMs on specific residues, please contact the Proteomics Core staff. We may be able to use targeted methods to specficially look for the peptide containing the PTM of interest.

Protein characterization

This service can include the following types of analyses:

* 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.

* Identification/confirmation of a mutated amino acid, dependent on location of mutated amino acid.

*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. Data is searched against a publicly available or customer provided protein database using either Mascot or Sequest search engines. Data is processed in Proteome Discoverer and Scaffold.

Additional proteases can be used, depending on protein sequence.

Proteomic profiling

Identification of proteins in a complex mixture, which can be submitted either in-gel or in-solution. Proteins are digested with typsin, then analyzed by LC-MS/MS using the QExactive HF or Lumos platform. Typically, a longer gradient is used to identify as many proteins as possible. Afterward, data is searched against a publicly available protein database using either Mascot, Sequest, or Andromeda search engines. Data is processed in Proteome Discoverer or MaxQuant. Depending on sample complexity, hundreds to thousands of proteins can be detected in a single analysis. This service is almost always coupled with quantitation to measure the relative changes in protein abundances between controls and samples.

This service can be coupled with TMT-based quantitation and high pH reversed phase peptide fractionation to maximize the number of peptide/protein identifications. Please contact the staff for details.

Phosphoproteomics profiling

Typical global phosphoproteomics workflow involves TMT-based quantitation, offline HPLC fractionation and High Select FeNTA enrichment. Phosphopeptides are analyzed using the Lumos platform and data are processed in Proteome Discoverer. Depending on sample complexity, hundreds to thousands of phosphopeptides can be detected in a single analysis.

 

Affinity purification-mass spectrometry (AP-MS) analysis

Identification of protein binding partners of an endogenous protein or tagged bait (protein, peptide, DNA, RNA, drug, etc) after affinity purification. 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 is searched against a publicly available protein database using either Mascot or Sequest search engines. Typically, data is processed in Proteome Discoverer and visualized in Scaffold (version 4).

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

Quantitation of peptides/proteins is typically performed to compare a control to a sample (ie, post-drug treatment, post-knockdown, etc). BIOLOGICAL REPLICATES ARE REQUIRED. Please contact the Proteomics Core staff to discuss experimental design. Types of quantitation include:

1. Stable Isotopic Labeling by Amino Acids in Cell Culture (SILAC). Customers are required to purchase SILAC reagents and perform the SILAC incorporation themselves. Prior to large-scale experiments, incorporation testing by LC/MS/MS analysis must be performed to determine the incorporation efficiency.

2. Isobaric tagging (TMT). The facility provides the labels and will perform the labeling step – up to 16 samples can be multiplexed.

3. Label-free using area under the curve or spectral counting. Each sample is analyzed individually. No additional sample preparation required.

4. Absolute quantitation using heavy labeled peptides for targeted analysis of specific peptides/proteins (see targeted analysis above).

Proteins are typically digested with trypsin then analyzed by LC-MS/MS. Peptide/proteins are identified and quantified using MaxQuant or Proteome Discoverer. Further data analysis is conducted in Persesus and/or R. The Proteomics Core can provide publication quality figures such as bar graphs, volcano plots, PCA plots, hierarchical clustering, as well as perform bioinformatics analyses using DAVID or IPA, among other programs

Typically performed on 908 Devices CE ZipChip coupled to the Thermo QExactive HF Biopharma. Protein or peptides are submitted lyophilized or in-solution, please contact us for buffer and concentration requirements. Intact molecular weight can be determined with high mass accuracy (ppm level) using one of our Orbitrap (high resolution) instruments. This service is used when high mass accuracy of a protein is needed, such as confirmation of a PTM or other modification.