FGCZ Intranet

The first step for the identification of unknown proteins is protein digestion using proteases (e.g. trypsin). We accept the following samples:

Coomassie Blue stained gel bands (whenever possible avoid silver staining)
in-solution (free of protease inhibitors!). Non-standard protocols may be required based on the buffer composition (please provide details in the order form). Limit the amount of SDS and avoid CHAPS/Tween.
beads (ideally magnetic ones). Please note that IgG/Protein A/.. would be extensively identified in your samples
for pull-down (affinity proteomics) experiments, check the Interaction Proteomics FAQ page

Depending on the type of samples:

Protein extracts/pull-down. Minimal recommended amount: 1 µg (less material can be used)
Confirmation of protein identity ("purified" protein). Minimal recommended amount: 50 – 250 fmol (optimal amount: 1 pmol)

For protein identification experiments, the results normally consist of:

Pdf Report with information on the used protocol and comment on the results
Scaffold file with all the results obtained using the Mascot search engine. See the data analysis sections for additional information
the results obtained using alternative search engines (e.g. PEAKS, Byonic, ...) are provided when needed (e.g., when customized databases are used)

Of course! Depending on the experimental design, we may use label-free or label-based methods

The characterization of all PTMs of one protein cannot be performed since they are often low abundant and require PTM-specific enrichment protocols. Moreover, searching the data for an unrestricted set of PTMs would lead to a large number of false-positive identifications.

PTMs such as phosphorylation, (di-)methylation, ubiquitination, acetylation MAY be identified without PTM enrichment, in case of previously enriched samples (e.g. pull-downs)
for large scale PTM studies an enrichment strategy should be discussed. FGCZ currently offers enrichment protocols for phosphorylation (TiO2, Ti4-IMAC) and, in special cases, acetylation (see here). Some of these analyses require the submission of a FGCZ project, please inquire.
the required amount of sample depends on the PTM. Rule of thumb: 10x more material is required compared to normal identification

The N- and C-Terminal protein identification depends on their protein physicochemical properties.

The simple analysis of intact proteins via MALDI-ISD (in-source fragmentation) may be successful for purified proteins.
for N-Terminal analysis, alternative methods based on N-Terminal labelling are implemented (e.g. dimethyl labelling)
for the large-scale identification of N-Termini (e.g. for identification of protease substrates) we do offer the approach TAILS

Depending on your biological question, we can support you with:

large scale (proteome-wide) relative protein quantification (mostly using Data-Independent Acquisition methods)
targeted protein analysis (analysis of a few protein targets over many samples)

We can support you with all the most common protein quantification methods:

label-free quantification (aka "LFQ", MS1-based): our standard approach for the large scale relative proteome quantifications. All details here
TMT labelling: for a small dataset (up to 16 samples). When combined with protein fractionation, it allows for a deeper protein coverage. All details here
DIA analysis: we normally use Data Independent Analysis methods for large datasets (100+) or, in some cases, for PTM analyses.
Other labelling methods: if you have SILAC samples, we can support you with the data acquisition and bioinformatic analysis.

We offer relative and, in certain cases, absolute protein quantification analyses. We select 3-5 proteotypic peptides for each protein and we monitor them using Parallel Reaction Monitoring (PRM) or SureQuant method approaches.
You can find more details here

Performing a preliminary QC test with 4 biological replicates will provide us information on:

the suitability of the protocol (feasibility, reproducibility)
what to expect (e.g. number of proteins, ...)
how to best design your experiment (e.g. number of replicates)

We normally estimate the sample size during the first QC experiment. If this is not possible, we suggest using at least 4 replicates for each condition. This number may dramatically change depending on the experimental design and the biological question.

Yes, we have already processed multiple orders with 100s of samples. We have automated sample preparation and high-throughput LC-MS methods (e.g., up to 200 samples per day). Nonetheless, we may not be able to support you depending on the type of samples or in case of tight timelines.

Please contact us before planning any type of LFQ experiment. The ideal starting material is 25 - 50 ug of proteins at a concentration of 0.2 - 1 ug/ul, but we can proceed with 1 - 2 ug and lower concentrations. The buffer should not contain detergents.

Yes, for protein quantification analyses we can extract the proteins from many tissue samples, please enquire.

Yes, we have established workflows for the analysis of FFPE samples from various origins, please enquire.

When possible, we perform protein digestion using the SP3 protocol or the PreOmics iST kit. In some cases, we use FASP, TCA or PCT digestion protocols.

We do not support single-cell proteomics, but we have established automated workflows for "low-input" proteomics. We can reliably quantify thousands of proteins from ~2000 sorted cell samples, but we can also analyse samples with only 500 cells or less. Check our dedicated Low-input analysis page

For DDA we mostly use FragPipe, MaxQuant, or at times PEAKS, Mascot, or Byonic
For DIA, depending on the experiment. we use DIA-NN, FragPipe-DIANN, or Spectronaut

The statistical analysis at FGCZ is mostly performed using our tool prolfqua: A Comprehensive R-Package for Proteomics Differential Expression Analysis
The package was published on J. Proteome Res. 2023, 22, 4, 1092–1104 (W. E. Wolski, P. Nanni, J. Grossmann, M. d’Errico, R. Schlapbach, and C. Panse, https://doi.org/10.1021/acs.jproteome.2c00441)

Please have a look at the following resources

Scaffold: Scaffold files (Proteome Software) can be used for the visualization of the protein and peptide identification results. Scaffold can be downloaded here: without a license, it woks as a Free Viewer.
- Quick FGCZ Scaffold Guide
- Official Scaffold User Guide (follow the link on the left)
PEAKS: click here for information on the software PEAKS and for the download of a free-viewer
Mascot: click here for the official tutorial videos from Matrix Science

One issue could be that the Memory is not sufficient. You can adapt the Memory under Edit>Preferences>Memory
Increase the Memory (eg 20000 MB) but be aware to not exceed the installed physical Memory (RAM) of your computer

Each experiment is different but the following software (particularly valuable for human and mouse) may be of interest

NextProt: Exploring the universe of human proteins
ProteomicsDB: expedite the identification of the human proteome
PaxDb: Protein Abundance Database

Check out the list tools for proteomics from Expasy. It contains dozens of interesting tools.
Do not forget to simply check Uniprot

Protein Identification

[+] Which type of samples can I send for protein identification analysis?

[+] How much material is needed for protein identification experiments?

[+] What will FGCZ provide as results of a protein identification experiment?

Post-translational modifications

[+] Do you offer large-scale phosphopeptides identification and quantification?

[+] Can you identify all the post-translational modifications (PTMs) on my protein? How much material is required for the analysis?

[+] Can you identify the N- and/or C-Terminal protein sequence(s)?

Protein Quantification

[+] Which type of protein quantification do you offer?

[+] How do you perform large scale proteome quantifications?

[+] How do you perform targeted proteome quantifications?

[+] Why do you always request a preliminary QC test, prior to any quantification experiment?

[+] How many replicates do you request for a relative quantification experiment?

[+] Can you analyse 100s of samples?

[+] How should we prepare the samples for a label-free quantification analysis (LFQ)?

[+] Do you accept tissue samples?

[+] Do you accept formalin-fixed, paraffin-embedded (FFPE) samples?

[+] Which is your protocol for protein digestion?

[+] Do you offer single-cell proteomics?

Data analysis

[+] Which software do you use for the analysis of DDA or DIA experiments?

[+] How do you perform the statistical analyses for your protein quantification experiments?

[+] How can I change some settings in Scaffold or PEAKS software?

[+] What can I do if I cant open the Scaffold file?

[+] Could you suggest some software for follow-up analysis of proteomics dataset?

[+] Could you suggest software that may help me studying my protein (e.g., visualization, computation, modifications, ...)?

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