CF Proteomics

The Core Facility Proteomics provides a full service from sample preparation (2) to data analysis and visualization (4) for the identification and quantification of proteins using mass spectrometry. We have experience with samples (1) from various sources and organisms and are equipped with state-of-the-art chromatography systems and mass spectrometers (3) that enable multiple LC-MS and LC-MS/MS experiments.

Workflow of a basic standard proteomics experiment in our facility. After we receive the samples (1), they are prepared (2) for the acquisition on our LC-MS/MS systems (3). The resulting data is then searched, analyzed and visualized (4, 5).

1. Samples:

types: primary cells, FACS sorted cells, protein and organelles IPs, proximity labelling (BioID, APEX), different tissues including Formalin Fixed and Paraffin Embedded (FFPE), etc. ...
organisms: E. coli, C. elegans, M. musculus, N. furzeri, H.sapiens, S.aureus etc.

2. Sample preparation:

digest: in-solution, on-beads or in-gel
desalting with OASIS solid phase extraction systems (Waters)
optional: high pH reversed phase fractionation
optional: phosphopeptide enrichment with AssayMAP Bravo (Agilent), ubiquitin and acetylation enrichment (kit)
optional: Automated BioID
optional: Low input samples with lysis and digestion using Covaris R230 (Covaris)
quantitation: label-free DIA or TMT

3. LC-MS/MS (key instruments):

Exploris 480 with FAIMS (Thermo)
Orbitrap Fusion Lumos Tribrid (Thermo)
Thermo Astral (Thermo)
LC system: nanoAcquity UPLC (Waters), M-Class UPLC (Waters), EvosepOne (Evosep) and Neo Vanquish (Thermo)

4. Search software:

Spectronaut (Biognosys) using Pulsar (Biognosys) (Bruderer et al. 2015)
MaxQuant (Freeware, Cox et al. 2008) using Andromeda (Freeware, Cox J et al. 2011)
Mascot server (Matrix Science) (Perkins et al. 1999)

5. Data analysis and visualisation:

in-house developed procedures build on R\Bioconductor
Spectronaut (Biognosys)
Fragpipe (Nesvilab)
DIA-NN (Demichev Lab) (Demichev et al. 2020)
Proteome Discoverer (Thermo)
SpectroDive (Biognosys)
SpectroMine (Biognosys)
Skyline (MacCoss Lab) (MacLean et al. 2010)

References

Cirri E, Knaudt H, Di Fraia D, Pömpner N, Rahnis N, Heinze I, Ori A, Dau T. Optimized automated workflow for BioID improves reproducibility and identification of protein-protein interactions. J Proteome Res. 2024 Sep 4. doi: 10.1021/acs.jproteome.4c00308.
Bartolome A, Heiby JC, Di Fraia D, Heinze I, Knaudt H, Spaeth E, Omrani O, Minetti A, Hofmann M, Kirkpatrick JM, Dau T, Ori A. Quantitative mapping of proteasome interactomes and substrates using ProteasomeID. Elife. 2024 Sep 4;13:RP93256. doi: 10.7554/eLife.93256.
Anitei M, Bruno F, Valkova C, Dau T, Cirri E, Mestres I, Calegari F, Kaether C. IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport. Cell Mol Life Sci. 2024 Aug 8;81(1):334. doi: 10.1007/s00018-024-05386-x
Di Sanzo* S, Spengler* K, Leheis A, Kirkpatrick JM, Rändler TL, Baldensperger T, Dau T, Henning C, Parca L, Marx C, Wang ZQ, Glomb MA, Ori** A, Heller** R.
Mapping protein carboxymethylation sites provides insights into their role in proteostasis and cell proliferation. Nat Commun 2021, 12(1), 6743 (** co-senior authors, * equal contribution).
Kelmer Sacramento, E. et al. Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation. Mol. Syst. Biol. 16, e9596 (2020).
Buczak, K. et al. Spatially resolved analysis of FFPE tissue proteomes by quantitative mass spectrometry. Nat. Protoc. 15, 2956-2979 (2020).
Gebert, N. et al. Region-specific proteome changes of the intestinal epithelium during aging and dietary restriction. Cell Rep. 31(4):107565 (2020).
Mackmull, M.-T. et al. Landscape of nuclear transport receptor cargo specificity. Mol. Syst. Biol. 13, 962 (2017)