Empowering research through mass spectrometry-based proteomics
Services
We provide a full service for your mass spectrometry-based proteomics experiment. This includes expert advice on your experimental design, sample preparation as well as processing and measuring of samples and data analysis including statistical analysis and data visualization.
For information on our services, browse our state-of-the-art analyses below. If you’re unsure whether we can support your experiment, please reach out — we’ll be able to provide you with more information.
In order to estimate the total price for a specific type of a proteomics experiment such as, for instance, a full proteome analysis, please contact the PCF (pcf@embl.de) to discuss.
Identification of purified proteins and protein complexes from in-gel or in-solution samples
We provide protein identification, verification of mutations, inserts, and PTM analysis of purified proteins and protein complexes in-gel or in-solution samples.
In-gel samples
Step-by-step overview of in-gel digestion: sample preparation, enzymatic digestion, and downstream processing.
We provide in-gel based analysis of proteins for:
Protein identification
Identification of PTMs
Identification of N- or C-terminal truncations
For this, we offer proteolysis by:
Specific enzymes such as trypsin, chymotrypsin, LysC, AspN, ArgC or GluC
Acid hydrolysis
Sample specifications
Load enough material to see a visible band of your protein(s) after Coomassie staining (typically 0.5-10 µg)
Send your sample as Coomassie-stained, excised gel band(s), see here for details
Ensure your Coomassie stain is MS-compatible
Do not microwave your gel
In-solution samples
Workflow of in-solution digestion: sample preparation, enzymatic digestion, and downstream processing.
We also provide in-solution processing of samples for identification of proteins and protein complexes.
Sample specifications
Please provide us with a maximum of 20 µg protein in 60 µL per sample
Analysis of post-translational modifications (PTMS)
We perform analysis of post-translational modifications (PTMs) either on level of purified proteins or – for selected PTMs – on a global scale.
PTM analysis on global scale
Overview of sample processing for analysis of PTMs on a global scale
For analysis of PTMs on a global scale, we provide support for quantitative analysis of:
Phosphorylation of serines and threonines (and tyrosines) via Fe-IMAC-based enrichment
Acetylation of lysines via antibody-based enrichment
Ubiquitination of lysines via antibody-based enrichment
Sample specifications
Please provide us with pelleted cells enough for ~ 500 µg of protein per sample
Please provide us with a minimum of 3 replicates per condition
In case you are limited in sample amount, please contact us to discuss your options
PTM analysis from purified proteins
For analysis of PTMs from purified proteins, please provide as with your sample from an SDS-PAGE as well as information on the nature of the modification(s).
We provide support in the analysis of protein-protein interactions of purified proteins and protein complexes using chemical crosslinking coupled to mass spectrometry (XL-MS).
Scheme of crosslinking coupled to mass spectrometry for purified proteins
At the moment, we provide XL-MS using the following crosslinkers
Disuccinimidyl phenyl phosphonic acid (DSPP or PhoX)
For this analysis, you will perform the crosslinking in your lab using our provided protocol up to the quenching step, then send us the sample for further processing. This ensures optimal experimental conditions during the crosslinking step and preserves sample integrity.
Sample specifications
Please provide us with a minimum protein amount of 50 µg per sample
The protein concentration of your sample should not be below 0.2 mg/mL
It is important that the sample buffer is free of primary amines as they would quench the crosslinking reaction
It is important that the sample is detergent-free
Data analysis
Data analysis
Our data processing and archiving workflow
Our support includes data analysis of your samples. Depending on your workflow, your data will be searched and analysed accordingly.
For all sample types, our data analysis includes
Database search
Spreadsheets with protein and peptide identifications and quantification data
Data visualization
Data discussion
Storage of raw data for 10 years
Upload of raw data on PRIDE repository for publications upon request
For more details on the different analyses and their deliveries, please read the information for the respective service below.
Protein identification
For protein identification, we will additionally provide sequence alignment plots and other descriptive visualizations. Data analysis for protein identification will be performed using MSFragger in the FragPipe suite using either a database provided by you or the respective Uniprot reference proteome.
Protein quantification
For protein quantification, we provide a comprehensive data analysis of the proteomics experiment along with the complete R script to ensure full reproducibility.
Our statistical pipeline includes
contaminant removal
correction for batch effects
variance-stabilising normalisation
if required – data imputation
Differential expression is assessed using moderated t-statistics from the limma package, and results are visualised through correlation plots and heat maps with clustering to highlight patterns in the data.
To support biological interpretation, we also provide a basic Gene Ontology enrichment analysis for standard model organisms.
For TMT, data analysis will be performed using MSFragger in the FragPipe suite using either a database provided by you or the respective Uniprot reference proteome. For DIA, data analysis will be performed using DIA-NN using either a database provided by you or the respective Uniprot reference proteome.
PTM analysis
For PTM analysis on a global scale, we provide a similar type of data analysis as for protein quantification data. Additionally, we include peptide-centric data and visualizations.
For PTM analysis from purified proteins, we will additionally provide sequence alignment plots and other descriptive visualizations.
For TMT and DDA, data analysis will be performed using MSFragger in the FragPipe suite using either a database provided by you or the respective Uniprot reference proteome. For DIA, data analysis will be performed using DIA-NN using either a database provided by you or the respective Uniprot reference proteome.
Crosslink analysis
For crosslinking, we provide spreadsheets containing all confidently identified crosslink peptides. This includes a datasheet that is ready for import to xiView where you can interactively investigate your data, visualize protein-protein interaction networks and map crosslinks on publicly available 3D structures.
Crosslink analysis will be performed using pLink3.
