In a kickoff meeting, we assess the feasibility of your project and decide together if mass-spectrometry is the best technology for answering your research question(s). In the rare case that gets more project applications than we can handle, we give priority to CeMM internal projects.
Please provide any relevant information about your project and enter it into iLAB. The more detailed your instructions are, the better we can help you. Please also upload a table which contains at least the following information:
Text written on samples
Sample type
Sample description, e.g. technical or biological replicate, condition, …
Chemical composition of buffer/solvent/… where proteins are dissolved. In the case samples are lyophilized, please indicate them and let us know if other chemical components are expected. This reduced the risk of low-quality samples or even sample losses.
Expected protein/peptide concentration or sample amount in the sample and how it was assessed.
Preferred injection order (of not provided, we will randomize your samples)
Risk information (according to §40 ASchG). Please indicate if samples have been inactivated and how.
Please provide information about the ethics approval if animal experiments are involved or human sample types are shipped.
Before sending or bringing any samples, please get in touch with Miriam Abele: mabele[at]cemm.oeaw.ac.at. Please store and ship your samples under appropriate conditions. We highly recommend shipping samples on Mondays or Tuesdays to ensure delivery during the week. Please always provide us with a tracking number or some reference so that we can monitor the status of your delivery. Once your samples have arrived at the MDP, we will notify you.
Our shipment address is:
Molecular Discovery Platform – Proteomics
Lazarettgasse 14, AKH BT 25.3, Floor 4, Room 04.G242
1090 Vienna, Austria
You will receive a mail once your samples have been measured and analyzed. We provide you:
Raw files
If you requested a data analysis, we are sending you a data analysis report including raw processed data, statistical analysis, and interactive plots.
Materials and methods
Acknowledgement sentence
After the project handover, we are continuously supporting you if questions arise.
Sufficient protein amount:
Rule of thumb: The more protein you have, the better it is to achieve the best results.
Concentrations in the range of 0.5 – 5 µg/µl are ideal.
LFQ single shot: >20 µg
LFQ fractionation: > 200 µg
TMT single shot: >20 µg
TMT fractionation: >50 µg
LFQ-based PTM enrichment: >2 mg
TMT-based PTM enrichment: >150 µg (for TMT18 = TMTpro) and >200 µg (for TMT11)
If you have less sample amount, please contact Miriam Abele. Ther might be possibilities to perform a proteomics experiment.
A fasta file: Bottum-up mass spectrometry relies on protein fasta files. Preferably, we accept fasta files from Uniprot (https://www.uniprot.org/proteomes), but can also work with NCBI downloaded protein fasta files or self-assembled fasta files. Please note that we can only identify proteins that are included in the fasta file. In case you have transgenic material, provide a fasta entry with these sequences as well.
Appropriate buffer composition: Although we can work with many different buffer and solvent systems, please refer to [link to buffer FAQ] or directly contact us.
We perform several quality control checks before and after your samples. If you run many samples, we will also monitor the machine status during the sequence.
Digested BSA: This low complex sample provides information about the chromatographic parameters of the system and also some hints about the mass spectrometer.
Pierce Hela (commercial standard): This high complex sample provides information about LC parameters and the mass spectrometer. We monitor the number of identifications, the total ion chromatogram shape and intensity, the retention time, mass accuracy, identification rates, and many more.
Blanks: We run blanks between samples or sample batches to quantify and reduce carryover.
Regular instrument calibrations: We calibrate our machines at regular time intervals to ensure the highest performance of our instruments. During calibrations, we monitor all parts of the instrument.
Project-specific quality checks: We check each acquired raw file and perform project-specific quality checks to guarantee the highest possible data quality.
No, we do not offer this service. Please get in contact with specialized facilities. Information can be found [pending]
It is essential to report the exact buffer composition when registering samples via iLAB. We accept proteins in the following lysis buffers:
8 M Urea
2% Sodium dodecylsulfate
2% Sodium deoxycholate
Additional buffer components which we accept, are:
Tris, pH range 7.0 – 9.0 (up to 100 mM)
HEPES, pH range 7.0 – 9-0 (up to 100 mM)
Ammonium bicarbonate, pH range 7.0 – 8.0 (up to 100 mM)
Dithiothreitol (up to 10 mM)
Tris(2-carboxyethyl)phosphine) = TCEP (up to 10 mM)
EDTA
In case your proteins are in another buffer or solvent or contain any other chemicals, please contact Miriam Abele.
In case you want to send us a sample at the peptide level, ideally send us your peptides lyophilized. Make sure that the sample does not contain any chemicals. We request the sample preparation protocol to evaluate if the sample is suited for MS.
Common contaminants are keratins originating from skin, hair, …. Peptides derived from keratins are masking other peptides from the sample during sample preparation. The consequence is lower peptide and protein coverage. Therefore, we recommend using clean tubes and containers and never touching gel, sample, etc with bare hands. For gels, we further recommend working in extra-clean areas.
Other common contaminants are BSA, residual proteins from cell culture media, for example FBS. Please wash your cells rigorously before sending samples.
Another source of contamination is polymers and detergents. They are both a serious threat to our sensitive instruments. Under any circumstances, please avoid sources of contamination. In the past, we experienced contamination arising from (membrane) filters, tubes, and pipet tips. In the case you were working with detergents, please indicate the use in the iLAB entry and we will act accordingly.
The number of proteins highly depends on your sample type, the organism under study, sample preparation, and the LC-ESI-MS/MS method. We are choosing the most suitable method for your individual needs.
There are several reasons why your protein of interest is not detected. They can be of biological or technical nature:
Protein is not expressed under the chosen conditions
Membrane proteins are harder to solubilize, therefore very tricky to detect with MS
Protein is low abundant
There are no MS-accessible peptides available, e.g. if the protease does not generate peptides of suitable length or if peptides have a bad MS-response factor
Peptides are modified and we do not search for them
Protein is not in the fasta file or mutations occurred
Proofing absence in proteomics is therefore very tricky and does not necessarily mean that the protein is not expressed.
