Our services are primarily available to EMBL researchers. We also support external academic institutions. Services for industrial partners may be offered depending on our available capacity.

To get the most out of your proteomics experiment, we strongly recommend reaching out to us as early as possible. Early contact allows us to support you in shaping the experimental design and ensuring optimal results.

We can assist with key aspects such as:

• Experimental design
  
• Determining the number of biological replicates
  
• Selecting appropriate controls
  
• Sample requirements (e.g., buffer conditions, sample volume)

To get started, please email us at pcf@embl.de.
Include the following information to help us assess your project and guide you effectively:

• Please provide your full name, address and group leader affiliation.
• Type of analysis (e.g., full proteome analysis, immunoprecipitations, proximity labelling, phosphoproteomics, protein identification from Coomassie-stained gels, secretome analysis).
• Sample type (e.g., cell line, primary cells, tissue, organs, animals)
• Availability of a FASTA database for your species:
  Databases can be found at: https://www.uniprot.org/proteomes
• Number and annotation of samples (e.g., wild-type, mutant, domain-only, treatments, time course) and number of biological replicates (a minimum of 3 biological replicates is required for statistical analysis)

By sharing this information, we can ensure that we design the most effective experiment tailored to your needs. This will also allow us to provide you with a rough cost estimate.

We can only accept samples accompanied by a completed service request form. This form provides all the essential information needed to process and analyze your samples.
A separate service request must be submitted for each individual set of samples.

Please note:

• Some sections of the form are only relevant to specific types of analysis. For example, gel images with annotated bands are not required for full proteome analysis.
• Protein identification relies on entries in a FASTA database. Overexpressed proteins from a different species will only be identified if they are included in the database.

If you are analyzing a custom or modified protein, please provide its full amino acid sequence, including:

• Linkers
  
• Cleavage sites
  
• Tags
  
• Any unnatural amino acids

To help us interpret the sequence, please highlight tags, mutations, and cleavage sites in color.

EMBL Internal users have to submit their service request form via the PPMS system. 

For this an account in the system is necessary. Please follow this link to access the system.

Please note that you cannot request a certain instrument and we do not allow pre-booking of instrument time.

These are the essential requirements for standard procedures:

Contaminants such as abundant serum proteins – especially from residual fetal calf serum (FCS) or bovine serum albumin (BSA) – can interfere with mass spectrometric analysis. To minimize these effects, we strongly recommend washing cultured cells three times with buffer such as PBS prior to lysis.

Most commonly used lysis buffers are compatible with our protein digestion workflow, including:

• Laemmli buffer (with or without bromophenol blue, β-mercaptoethanol, or dithiothreitol)
• SDS sample buffer
• RIPA buffer
• Others

Our in-solution tryptic digestion is based on a modified SP3 protocol (PMID: 25358341). For a detailed list of tested and compatible reagents, please refer to Figure 1B in the referenced publication.

You may include EDTA-free protease inhibitor cocktails or phosphatase inhibitors in your lysis buffers, as they are compatible with our workflow.

If cell lysis results in the release of genomic DNA, the sample may become highly viscous. To reduce viscosity and ensure proper processing, please treat samples with benzonase or perform sonication. This step is also important for efficient recovery of nucleic acid-bound proteins.

Accurate protein quantification is essential for reliable mass spectrometry-based relative quantification. To ensure consistency and comparability across your samples, we require:

• Equal protein input for each condition and biological replicate
  
• Accurate protein concentration measurement, ideally using a BSA standard curve to allow both relative and absolute quantification

To determine protein concentration reliably, we recommend the following approaches:

• Tryptophan Assay
  Based on the method described by Wiśniewski and Gaugaz (DOI: 10.1021/ac504689z), this fluorescence-based assay is cost-effective, user-friendly, and compatible with a wide range of sample types and buffer components.
  Note: This assay requires black microplates and a fluorescence-capable plate reader. Please check compatibility with your equipment.
  
• Colorimetric Assays
  • Bradford assay
  • BCA (Bicinchoninic Acid) assay
    These are widely used and generally reliable for protein concentration determination.

Important Note
Protein concentration measurements using NanoDrop or similar spectrophotometers are not reliable and should be avoided.

In general, we recommend performing all possible quality control steps to ensure the success of the mass spectrometry analysis of your samples. These steps may include protein quantification, SDS-PAGE combined with Coomassie or silver staining, and Western blot analyses to verify knockout, expression, or enrichment of the protein of interest. However, we understand that sometimes it is necessary to proceed with mass spectrometry analysis without controlling every step, for example, when working with limited sample material.

If you plan to submit samples at the peptide level, please contact us in advance. Note that we can only accept peptide samples prepared in mass spectrometry-compatible buffers.

For a full proteome analysis, we recommend using buffers with harsh detergents such as SDS to ensure complete cell lysis. A suitable choice is RIPA buffer (0.1% SDS, 1% deoxycholate, 1% NP-40, 150 mM NaCl in 50 mM Tris/HCl, pH 7–8), supplemented with EDTA-free protease inhibitors. To reduce sample viscosity and facilitate protein extraction, it is essential to degrade DNA by adding Benzonase or using sonication, followed by removal of cell debris via centrifugation.
Accurate protein quantification is critical to provide equal protein amounts for reliable quantitative comparison between different conditions. We recommend determining protein concentration using BCA, Bradford, or Tryptophan assays, as methods like NanoDrop are not sufficiently accurate. Please adjust all samples to 20 µg of protein in 60 µL of lysis buffer (e.g., Laemmli buffer). Insufficient protein amounts may lead to incomplete proteomic analysis, while excessive protein concentrations can reduce digestion and TMT labeling efficiency.

If you cannot provide 20 µg per sample, please contact us to discuss alternatives.
For further details, please also see the Sample Submission section.

• Please do not perform protein quantification on eluates from IP or proximity labeling experiments. Instead, submit equal elution volumes for all samples, providing no more than 60 µL of eluate per sample.
  
• Ensure that all beads are completely removed before submission.
  
• Use an equal number of cells or equivalent protein amounts as starting material for each sample.

To isolate protein complexes, buffers containing 0.1% Triton X-100 with 150 mM NaCl in 100 mM HEPES/NaOH (pH ~7.5) are commonly used as a good starting point. However, we recommend optimizing lysis conditions for your specific protein of interest. More detailed guidance on lysis and immunoaffinity purification can be found here.

Special considerations

• Immunoaffinity purification of nuclear proteins can be challenging due to the harsh lysis conditions required. For enrichment of nuclear protein complexes, we recommend proximity labeling approaches.
  
• Please note that sonication may affect the integrity of protein complexes and should be used with caution.

Quality Control Recommendations

• Perform a Western blot to verify enrichment of your protein of interest and to check for co-immunoprecipitation of known interaction partners.
  
• Run SDS-PAGE followed by Coomassie or silver staining depending on sample amount. Confirm that control samples contain proteins and that the bait-IP sample shows reduced complexity compared to the input lysate.
  
• Elution with Laemmli buffer is generally effective for releasing protein complexes.
  
• We do not recommend using formaldehyde-based crosslinking.

For secretome analysis, it is essential to culture cells in serum-free medium. The presence of abundant proteins, such as those from fetal calf/bovine serum, can dominate the sample and produce prominent peptides. Since the mass spectrometer prioritizes analyzing the most abundant peptides first, this can lead to detecting serum proteins like albumin instead of the proteins of interest in your sample.

We provide services for the mass spectrometric analysis of extracellular matrices (ECMs). Below is a selection of key publications relevant to ECM proteomics and sample preparation:

• Khan MM, Galea G, Jung J, et al. Dextromethorphan inhibits collagen and collagen-like cargo secretion to ameliorate lung fibrosis. Science Translational Medicine. 2024 Dec;16(778):eadj3087. DOI: 10.1126/scitranslmed.adj3087. PMID: 39693409.
  
• Naba A, Clauser KR, Hynes RO. Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis. J Vis Exp. 2015;(101):e53057. DOI: 10.3791/53057. PMID: 26273955.
  
• McCabe MC, Schmitt LR, Hill RC, et al. Evaluation and Refinement of Sample Preparation Methods for Extracellular Matrix Proteome Coverage. Mol Cell Proteomics. 2021 Jan;20:100079. DOI: 10.1016/j.mcpro.2021.100079. PMID: 33845168.

Phosphoproteomic analysis requires a high amount of input material—typically around 500 µg of total protein. The number of phosphopeptides detected is strongly dependent on the amount of starting material provided.

To ensure optimal results, please contact us in advance to receive our lysis protocol, which is specifically optimized for compatibility with our downstream phosphopeptide enrichment workflow.

Plasma and serum contain a few very high-abundance proteins (e.g., albumin, immunoglobulins) that can interfere with the in-depth analysis of lower-abundance proteins by mass spectrometry.

To improve proteome coverage, we recommend using the ENRICH-iST kit from PreOmics, which is specifically designed for the depletion of abundant plasma proteins: https://www.preomics.com/products/enrich-ist

• Please send only cut gel bands from Coomassie-stained gels.
  
• Ensure your Coomassie stain is MS-compatible by consulting the manufacturer’s datasheet or contacting their customer service.
  
• Follow the instructions outlined in our gel-cutting procedure.
  
• Gel images are mandatory; samples without an accompanying gel image will not be accepted.
  
• Clearly label the protein ladder and number each cut band.
  
• Mark the exact area excised from the gel.
  
• Include empty lanes between samples and avoid overloading.
  
• Note that the quality of your gel directly affects the accuracy of MS analysis results.

• Wear gloves at all times
  • Always wear gloves during sample preparation to prevent introducing contaminants, such as human keratins, into your samples. Ensure gloves are changed regularly.
• Use proper sample handling and storage conditions
  • Store samples in conditions that preserve their integrity for mass spectrometric analysis. If freezing is required, use appropriate vials and store at -80°C or lower, depending on your sample’s needs.
• Label samples clearly
  • Ensure that all samples are clearly labeled with your initials and single consecutive numbers (avoid detailed explanations on the tubes). Detailed explanations should be provided in our service request form.

• Use fresh reagents for buffer preparation
  • Always prepare buffers using fresh stocks and chemicals. Older chemicals may be a source of cross-contamination or keratin contamination. Additionally, some chemicals degrade, oxidize, or attract moisture over time, which can affect the quality and consistency of your samples.
• Clean equipment regularly
  • Clean all tools, surfaces, and equipment thoroughly with 70% ethanol before and after use. Use lint-free wipes (e.g., Kimberly-Clark Kimtech Science) to ensure no fibers or debris are left behind.

For gel samples only:

• Use clean containers for gels
• Cover with a lid
  • Ensure that containers used for storing gels are always sealed with a lid to prevent contamination and exposure to airborne particles.
• Prepare gel bands on clean surfaces

• Contact Us First: Do not send samples without prior discussion with PCF staff.
  
• Packaging: Use secure, leak-proof tubes. Do not use parafilm. Clearly label each tube with your initials and a sequential number.
  
• Sample Preparation: Spin down and freeze all protein samples. Place all tubes into a single container (e.g., a box or bag) inside the styrofoam shipping box. This ensures all samples remain together and protected during transit.
  
• Documentation: Include a printed copy of the completed service request form in a transparent sleeve inside the shipment. (Example image)
  The form must list all treatments applied to each sample, in the same order as the samples are arranged in the box.

• Ship to the following address:

• Timing: Avoid shipping during German public holidays. Ship early in the week to ensure samples arrive before the weekend. Do not ship over the weekend.
  
• Notification: Inform PCF staff once samples are sent. Email a digital copy of the service request form to pcf@embl.de.

Additional Information for shipments from non-EU countries

In order to avoid any delay in receiving your samples, please follow our requirements for shipping samples from non-EU countries and add a proforma invoice to your shipment.

• In-gel samples are processed weekly, typically on Thursdays. Gels received by Wednesday evening will be included in that week’s run. If no technical issues arise, results can usually be expected the following week.
• In-solution samples have a standard turnaround time of 4 to 6 weeks from the date of receipt.

Please note: During periods of high demand, processing times may be longer than stated.

Please note that upon completion of the project (after receipt of the data) we will store the remaining samples for 8 weeks.

Please also note that we do not send any samples back.

The raw files of your experiment are archived for 10 years on our server.

• Internal rates apply to current EMBL members and active EMBO Young Investigator / EMBO Installation Grantee / EMBO Global Investigator.

• External rates (as per our published price list) apply to academic institutions and former EMBL members.

• We do not offer discounts for higher sample numbers.
  
• Projects are generally not offered on a collaborative basis only, as cost recovery for consumables and instrument maintenance is required.
  Please note: All experiments, including pilot studies, must be fully compensated.
  
• Industry partners are subject to special pricing.

Please note: Our facility is charging solely for the consumables and measurement time. Costs depend on the sample type, complexity and number of samples.

These are our current fees for external customers (as of August 2025):

We deliver a comprehensive data analysis of the proteomics experiment along with the complete R script to ensure full reproducibility. Our pipeline includes contaminant removal, correction for batch effects, variance-stabilising normalisation and – 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 data analysis, we primarily use UniProt Proteome Databases, which include only one representative entry per gene. As a result, information on individual isoforms is not reported.

Please consider whether this level of analysis is suitable for your research question. If distinguishing between isoforms is important for your study, let us know in advance so we can discuss possible options.

A full proteome analysis of an immortalized cell line, such as HEK293T cells, typically yields around 8,000 identified protein groups (with a minimum of 1 peptide per protein), of which approximately 7,000 proteins are quantified (minimum 2 peptides per protein) across compared conditions.

For other cell types, such as Escherichia coli, we generally identify about 2,200 proteins, with around 2,000 proteins quantified. These numbers vary significantly depending on sample type and proteome complexity.

For samples like tissue lysates, the number of quantified proteins is typically lower, around 4,000. Protein identification and quantification also correlate with MS analysis time and sample quality, including factors like lysis conditions and contamination.

For protein complex analyses (e.g., immunoprecipitations or proximity labeling), only a subset of the proteome is expected—often a few hundred proteins in a typical 90-minute LC-MS/MS run. We typically use TMT-based quantification for these experiments and to improve the quantification precision and achieve deeper interactome coverage, we usually perform high-pH offline fractionation, analyzing multiple fractions (commonly six).

The total number of identified proteins also depends on the quality of the FASTA database used. Please note, we cannot identify proteins absent from the database.

In case your protein of interest is not detected, there are several possible reasons. Smaller proteins tend to produce fewer tryptic peptides. The distribution of tryptic cleavage sites along the protein sequence may be uneven. Low expression levels can also limit detection. Additionally, the protein’s cellular localization and the efficiency of lysis can affect whether it is identified. Finally, proteins expressed from other species may not be detected.