Proteomics

An Introduction

The NBCC provides full support for your proteomics needs from initial study concept to manuscript preparation and data submission to public repositories. The facility uses bottom-up proteomics (shotgun sequencing) to identify peptides using mass spectrometry and then matches them back to known proteins. The facility has developed and validated a first-in-class pipeline for protein-protein interaction studies that culminates in publication-ready visualizations. In addition, we provide advanced instrumentation and in-depth expertise for differential proteome abundance and chemical proteomics studies.

Brendon Installing Flex Source

Quick Navigation

Services

DDAvsDIA

1. Mass Spectrometry Acquisition

We perform 2 main modes of mass spectrometry acquisition, but also offer labelled and targeted quantitative analysis. Users can submit already processed peptide solutions and opt for one of the following acquisition modes:

Data-dependent acquisition (DDA) fragments the top N (typically 10) most abundant precursors in each MS1 scan window. This allows for a confident matching of fragmentation pattern (to determine peptide sequence) to the m/z (mass over charge). The MS1 scan detects all prescursors/peptides present and reports their mass and charge. DDA results are easy to analyze but are often biased and stochastic because it selects and fragments the most abundant precursors. DDA is suggested for simple mixtures (100 proteins or less), complex post-translation modifications studies, and de novo sequencing (not offered by the facility). DDA is also our standard for protein-protein interaction (PPI) studies. DDA results through our facility are reported as spectral counts (number of times a fragmentation pattern is matched to a peptide that is matched to a protein).

Data-independent acquisition (DIA) is an unbiased approach which breaks up the MS1 scan window into smaller windows of sizes 50 m/z or below. All the precursors in each of these small windows are fragmented together. This produces a complex spectra which may contain fragmentation patterns from multiple precursors. DIA data needs to be analyzed with complex computational algorithms that require more computational power than DDA. DIA is recommended for complex protein mixtures (5000+ proteins) and simple post-translational modifications (phosphorylation or ubiquitin remnant after trypsin cleavage (GG)).

We offer services to label peptide samples with tandem mass tags (TMT) to multiplex samples for quantification, up to 18-plex, in one mass spectrometry acquisition run. TMT multiplexed samples will be analyzed on our Thermo mass spectrometers. We also offer services to quantify samples labelled with other modalities such as SILAC and iTRAQ. Please contact us to discuss your project and the most appropriate methodology.

For quantification of a specific set of proteins, peptides or post-translational modifications, we offer targeted quantification analysis on the Fusion Tribrid Lumos through parallel reaction monitoring (PRM). The costing will be determined after the project is defined and will include all assay setup costs.

2. Protein-Protein Interaction (PPI) or Proximal Protein Mapping

We are specialists in protein-protein interaction (PPI) mapping. We offer reagents, expertise, and a data analysis pipeline to identify interacting proteins (immunoaffinity purification (e.g., FLAG tag)) and proximal proteins (proximity-dependent biotinylation (PDB) methods (often referred to as BioID)). These assays can be performed re-iteratively for proteins in the same pathway or protein family to create maps of signaling networks. See resources for reagents and protocols for sample preparation.

Protein-protein interaction
DPA

3. Differential Proteome Abundance

We perform sample processing and global proteome analysis to determine differences between cell states. We comprehensively track changes between different populations (e.g., normal vs disease) or treatments. To achieve high protein coverage, we identify and quantify proteins from these complex mixtures in an unbiased manner on our state-of-the-art instruments (e.g., timsTOF Pro 2 or Orbitrap Astral).

4. Chemical Proteomics

Assessing target engagement is a key step in the drug development process. We offer pull-downs and competition assays to identify which proteins bind small molecules or biologics. These tests enable the rapid assessment of on- and off-target binding during early-stage drug validation.

Chem Proteomics
Data Analysis

5. Data Analysis and Visualization

We have created resources to help manage, analyze, and visualize protein-protein interaction data. For protein-protein interaction (PPI) studies, we have a complete pipeline going from storage and management of data with ProHits, searching of data with MSFragger, analysis of PPI data with SAINTexpress, and visualization with ProHits-viz.

ProHits

ProHits is a laboratory information management system (LIMS) that manages and annotates data. MSFragger and SAINTexpress are integrated for a complete analysis pipeline.

ProHits-viz

ProHits-viz is an online visualization tool for the seamless generation of heatmaps and dot plots from SAINTexpress results. Results can be exported for Cytoscape interaction mapping.

Cell-Map

Cell-Map is a resource of proximity-dependent biotinylation data from 192 subcellular markers in HEK293 cells. This is an online tool that can be used for localization analysis of proximity-dependent biotinylation data.

Equipment

The NBCC provides proteomics services using 5 mass spectrometers from 3 different vendors: ThermoFisher Scientific, Bruker, and Sciex. All instruments can perform DDA and DIA.

ThermoFisher Scientific

All Thermo instruments are compatible with quantitative approaches such as parallel-reaction monitoring (PRM), data-independent acquisition (DIA), and multiplexing using isobaric tandem mass tags (TMT).

Orbitrap Astral

The Orbitrap Astral excels when the highest degree of proteome coverage and sensitivity is required. It enables the identification of 8000 proteins from a single sample with a throughput of 80 samples per day.

Fusion Tribrid Lumos

The Fusion Tribrid Lumos offers a variety of workflows, including confident identification of complex post-translational modifications and the ability to tease apart complex cross-linked mixtures. The Lumos’ capabilities are supported by electron transfer dissociation (ETD) and MSn fragmentation.

Orbitrap Astral

Bruker

Bruker mass spectrometers coupled to Evosep Ones (HPLCs) enable robust and fast acquisition of samples by utilizing trapped ion mobility technology coupled to parallel accumulation serial fragmentation (PASEF).

timsTOF Pro 2

The timsTOF Pro 2 is our workhorse for standard workflows such as protein-protein interaction and global proteome studies, enabling the identification of 6000 proteins per sample at a rate of 30 samples per day.

timsTOF SCP

The timsTOF SCP is a single cell proteomics solution, enabling detection of 4000 proteins from a single-cell level sample (250 pg) and can process 40 samples per day.

Sciex

TripleTOF 6600

The Sciex TripleTOF 6600 is geared towards the identification of protein complexes and protein-protein interactions.

Pricing

These prices are for academic laboratories and are guidelines only. Interested users should contact Cassandra Wong (cwong@lunenfeld.ca) to discuss their project and receive a quotation (including volume discounts).

Sample Preparation Costs (per sample, minimum of four samples)
Service Internal Academic Cost External Academic Cost
Affinity Purification: FLAG or other tag $ 120 $ 130
Affinity Purification: Proximity-dependent biotinylation $ 135 $ 150
Lysate preparation and trypsin digest $ 120 $ 140
Phosphopeptide enrichment $ 180 $ 200
In-solution trypsin digest $ 50 $ 55
Mass Spectrometry Costs per Sample
Service Internal Academic Cost External Academic Cost
Identification (DDA) or Quantification (DIA) - AP-MS or BioID $ 110 $ 120
Quantification (DIA) - cell lysate or cell fractionation Starting $ 135 Starting $ 150
Quantification - DDA with TMT Starting $ 360 Starting $ 380

Customized mass spectrometry analyses are also available.

All services include initial consultation and project planning with a proteomics manager, database searching including SAINTexpress for protein interactions, full report and data consultation. For additional data analysis, the fee is $90/hour (internal) and $99/hour (external).

Protocols

Protocols

Name PDF
NBCC PR1 In-gel digest
NBCC PR2 FLAG-IP
NBCC PR3 BioID Flp-In T-REx cell prep
NBCC PR4 BioID lentiviral cell prep
NBCC PR5 BioID affinity purification

Note: For miniTurbo, TurboID, and ultraID, recommended biotin labeling time is 1 hour.

Policies

General Policies

  • Hours of Operation are 8 am to 4 pm Monday to Friday. Please contact Cassandra Wong (cwong@lunenfeld.ca) to discuss your project before dropping off any samples. A sample intake sheet must be completed for every submission.

  • Samples should only be shipped on Monday or Tuesday to ensure samples arrive before the weekend. Please ensure there is sufficient dry ice to account for any shipping delays. Ship samples to “Cassandra Wong, Mount Sinai Hospital, 600 University Ave, Room 968, Toronto, ON, M5G 1X5” and send tracking information to Cassandra Wong (cwong@lunenfeld.ca).

    • Acknowledgement

    Preferred acknowledgment for the Network Biology Collaborative Centre Proteomics Facility:

    The authors wish to thank [staff name] of the Network Biology Collaborative Centre Proteomics Facility (RRID: SCR_025375) at the Lunenfeld-Tanenbaum Research Institute for [service]. The facility is supported by the Canada Foundation for Innovation and the Ontario Government.

    Click here for our publication policy and preferred acknowledgement for other NBCC Facilities.

    NBCC

    Network Biology Collaborative Centre

    Sinai Health, Lunenfeld-Tanenbaum Research Institute
    600 University Avenue, Room 970
    Toronto, Ontario Canada M5G 1X5

    Facilities
    More
    Socials

    2024 Network Biology Collaborative Centre, Lunenfeld-Tanenbaum Research Institute, Sinai Health System