Mass Spectrometry Service
The Mass Spectrometry group performs small to large scale proteomic and metabolomic analyses for all research groups at the MPIMG. We provide a versatile proteomics and metabolomics platform for the institute including sample preparation, sample digestion, mass spectrometry, and data analysis.
Before you start a project
As there is no universal proteomics/metabolomics workflow, and experiment design and sample preparation are very important, it is mandatory to contact David Meierhofer for consulting before you start a project.
Our expertise includes:
- Identification and quantification of single proteins
- Identification and quantification of proteins in complex proteomes (Labelled- and label-free)
- Quantitative analysis of post-translational modifications of proteins (e.g. phosphorylation, ubiquitination, acetylation, methylation…, which require peptide enrichments)
- Protein pull-downs and interaction studies
- Identification and quantification of single metabolites
- Identification and quantification of metabolites in complex proteomes
The MS facility includes two mass spectrometers, which are used in dependence on the sample requirements:
- QExactive HF, hybrid quadrupole-Orbitrap (Thermo Scientific)
- This instrument is set up to quantitatively analyze peptides proteome wide. In contrast to the QTrap, you don’t need to know your proteins and PTM´s (shotgun proteomics).
Proteomics: We developed a bunch of pipelines in order to decipher entire proteomes and post translational modifications, as well as single proteins, e.g. bands from SDS page gels.
We are able to detect and quantify several thousand proteins in a proteome. As phosphorylation’s are of main interest, we can now detect more than 15.000 individual phosphorylation sites / proteome with our enrichment method.
We can analyze a proteome in one run (1D), or make several fractionation steps (gel bands, SAX or SCX separations) for a 2D proteome with a much higher identification rate.
For relative quantification, we are using SILAC (stable isotope labeling by amino acids in cell culture) or label free quantification (LFQ).
- QTrap 6500, linear ion trap (Sciex)
- This instrument is best for targeted analysis of known peptides or metabolites. For each peptide or metabolite, an individual method (MRM) has to be established on the instrument. It is the most sensitive (low atto mol range) mass spectrometer in house.
Metabolomics: we created about 400 MRM methods to analyze metabolites, including key cellular components such as amino acids, acylcarnitines, bile acids, carbohydrates, vitamins, hormones, nucleotides, biogenic amines, ceramides, sphingomyelines.... .
Not all compounds are well detectable by mass spectrometry, for example due to weak ionization rates. Therefore, derivatization can be applied to metabolites which are not easily ionized. For example, HCl-butanol can be used to derivate carboxylic acids.
- We use several bioinformatic tools to analyze large proteomic datasets such as MaxQuant (search engine: ANDROMEDA) in conjunction with Perseus for statistical analysis (http://maxquant.org/), or MultiQuant for metabolome data.
- Pathway analysis of differentially expressed proteins can be further explored with bioinformatic tools such as Gene Set Enrichment Analysis (GSEA) (Subramanian, et al. 2005), IMPaLa (Kamburov, et al. 2011), and ConsensusPathDB (Kamburov, et al. 2013).
- Protein-protein interaction (PPI) network analysis and identification of hub proteins can be done by programs such as String (Franceschini, et al. 2013) and Cytoscape (Cline, et al. 2007).
- We usually send out excel sheets of the analyzed samples. All data is archived on local servers and researchers can have access to raw data upon request, e.g., we usually upload the original MS files to depositories and make them public as soon as the manuscript is accepted (on request).
Things to avoid
Mass spectrometry (MS) uses very sensitive mass spectrometers and not all chemicals are MS compatible. Some can harm the instrument, e.g. corrosive chemicals; others induce ion suppression, leading to dwarfed or no peaks. Detergents and polymers are the main source of ion suppression and have to be avoided, even the smallest traces. Usually, detergents cannot be washed out of samples completely. For most of the protocols, there are mass spectrometry compatible versions. Some buffers even contain undeclared substances. Other chemicals can modify peptides and introduce artificial modifications and should therefore be avoided, (e.g. methanol can introduce artificial methylation sites).
Below is a list of compatible/incompatible detergents for in-solution or in-gel digestions.
In-solution Digestion
Compatible Detergents:
0.05%-1% SDS
0.05%-0.5% CHAPS
Incompatible Detergents:
Nonidet P-40 (which can no longer be purchased; Sigma is substituting CA-Igepal 630)
Triton® X-100 (or any derivative)
Igepal/PEG (any derivative)
Brij®-35 (or any derivative)
Tween®-20
OTG
CHAPSO
Type NP40/NP40 alternative
In-gel Digestion
Compatible Detergents:
SDS (up to 2%)
CHAPS (up to 4%)
Nonidet P-40 (up to 1%); which can no longer be purchased; Sigma is substituting CA-Igepal 630
Incompatible Detergents:
Triton® X-100 (or any derivative)
Tween®-20
Igepal/PEG (any derivative)
Brij®-35 (or any derivative)
OTG
CHAPSO
Type NP40/NP40 alternative
Some detergents can be separated from the sample by standard SDS-PAGE or acetone precipitation. However, Triton-X and Tween-20 cannot be used under any circumstances. These cannot be removed from your sample using dilution, washing, detergent spin columns, or SDS-PAGE.
General guidelines for protein extractions:
- Work in a clean environment, protect samples from skin /wool keratin and dust.
- Wear gloves, close tubes, tip boxes after use.
- Keep sample storage and processing time as short as possible.
- Keep sample on ice if non-denaturing buffer is used and use protease/phosphatase inhibitors.
- Use LoProtein binding tubes/plastic ware.
- As some proteins/peptides bind to glass/plastic ware, use as less protocol steps as possible.
- Measure protein content (usually 10- 50 µg of protein extract is required, for PTM.
- Avoid longer storage of samples in fridge, freeze (-80°C) proteins if it needs to be stored for more than a day.
- Avoid any kind of detergents (e.g. NP-40).
Publication policy
For collaborative projects, any intellectual input from our scientific members into the project during planning, execution and analysis of the experiments justifies a co-authorship on a manuscript that contains data generated in our facility.