Service

The mass spectrometry module of the CFH (Core Facility Hohenheim) offers consultation for mass spectrometry analysis of proteins and metabolites to discuss your scientific issues. Consultation comprises individual requirements for your research projects such as suitable sample preparation and mass spectrometry methods as well as questions concerning prices and time frame. Please arrange an individual appointment for a consultation by e-mail or phone. You can find information about charges for mass spectrometry service analyses in the current price list.

Mass Spectrometry

The CFH offers the following mass spectrometry services:

Molecular Weight Determination (MALDI-TOF, LC-ESI-MS)

For molecular weight determination of peptides and proteins by MALDI-TOF or LC-ESI-MS samples can be submitted in lyophilized form or in solution. Please read the information for sample preparation for mass spectrometry analysis first (see FAQ).

Peptide Mass Fingerprinting (PMF, MALDI-TOF)

Proteins from 1D-, 2D- and 2D DIGE-gels can be identified by peptide mass fingerprinting (PMF). Please use staining methods for your gels that are compatible with the subsequent MS analysis (see FAQ). Gels should be submitted at the CFH for PMF analysis together with the sample submission sheet and a picture of the gel. Please indicate the bands/spots that should be analysed by PMF on the picture. The PMF analysis includes an in gel digest with trypsin (other proteases on request), concentration and desalting using ZipTips, MALDI-TOF analysis and a database search (Mascot, Sequest or Andromeda). Moreover, a verification of the PMF result by a fragmentation spectrum (MS/MS) of one peptide is included.

Identification and quantification of proteins (nanoLC-ESI-MS/MS)

For the identification of proteins from complex mixtures two high resolution FT-ESI-MS/MS mass spectrometers (LTQ-Orbitrap XL, Q-Exactive Plus, Thermo Fisher Scientific) as well as nano-UHPLC-systems (Easy-nLC 1200 or EasynLC 1000, Thermo Fisher Scientific) are available at the CFH. The high sensitivity and mass accuracy of the FT-ESI-MS/MS mass spectrometers facilitates identification and quantification of proteins. For identification and quantification of proteins in samples of low or medium complexity (≤ 1000 proteins) HPLC gradients ≤ 2 h are usually sufficient. Identification and relative quantification of proteins in highly complex mixtures including several thousands of proteins (“proteomics”) often requires HPLC gradients ≥ 2 h and a mass spectrometer with high mass accuracy and high scan speed. For these experiments a Q-Exactive Plus mass spectrometer is available at the CFH that has higher scan speed and higher mass accuracy of MS/MS spectra compared to the Orbitrap XL, leading to a higher number of identified and quantified proteins in highly complex samples.

Identification of proteins following mass spectrometry analysis can be performed with different database search algorithms (Mascot, Sequest, Andromeda). For statistical analysis of proteomics data additional software packages are available (MaxQuant 1.5.5.1, Perseus 1.5.5.3, Progenesis LCMS 4.1.4). The costs for enzymatic digest of the sample, mass spectrometry analysis and the database search are covered by the price for the analysis.

Analysis of posttranslational protein modifications (nanoLC-ESI-MS/MS)

Identification and quantification of posttranslational protein modifications by mass spectrometry sometimes requires chromatographic separation of samples in combination with specific enrichment of modified peptides. Therefore projects aiming at analysis of posttranslational modifications should be discussed with the team of the CFH in advance. Protocols for specific enrichment of modified peptides are already established (phosphorylation, N-glycosylation) at the CFH or can be established on demand.

ESI-MS/MS is better suited for the analysis of posttranslational protein modifications than MALDI-TOF since electrospray ionization can be directly coupled to a liquid chromatography separation. Therefore the mass spectrometer can identify more peptides per protein which makes the identification of modified peptides much more likely. For the analysis of modified peptides two highly sensitive nano-LC FT-ESI-MS/MS-systems (Q-Exactive Plus, LTQ-Orbitrap XL, Thermo Fisher Scientific) are available at the CFH. The costs for enzymatic digest of the sample, mass spectrometry analysis and the database search are covered by price for the analysis.

Targeted quantification of proteins by PRM (nanoLC-ESI-MS/MS)

Targeted quantification of proteins can be performed on the Q-Exactive Plus mass spectrometer using PRM (Parallel Reaction Monitoring). Using PRM target peptides can be detected with similar sensitivity than in a SRM (Selected Reaction Monitoring) experiment. In contrast to SRM experiments all fragment ions of a given target peptide are recorded in parallel in a single high resolution scan. For a PRM experiment only the m/z ratio of the target peptide(s) must be known, which makes the experimental design easier compared to SRM experiments.

To set up a PRM experiment we need to know which proteins should be quantified. The selection of suitable target peptides can be performed based on a theoretical digest of the target protein. Which peptides and fragment ions are finally used for quantification of the target protein, can be decided post acquisition based on the mass spectrometry data. Absolute quantification is also possible, but requires a calibration curve. For this purpose the target peptide(s) are needed as standard substance(s) (e.g. by peptide synthesis).

Metabolites & Metabolomics

Mass spectrometry analysis of different metabolites (e.g. lipids, carbohydrates, polyphenols, etc.) of different origin (e.g. plant material, cell culture, animal tissue, etc.) requires an individual sample preparation in each case. Therefore sample preparation for mass spectrometry analysis of metabolites has to be performed by the users themselves. The CFH team can help to establish suitable protocols for sample preparation.

Molecular Weight Determination (MALDI-TOF, ESI-MS, LC-ESI-MS)

For a high resolution molecular weight determination of small organic molecules (metabolites, etc.) by MALDI-TOF or ESI-MS samples can be passed in solution or freeze-dried. Please first read the instructions for sample preparation for mass spectrometry analysis [[see FAQ]]. Samples that have already been purified by HPLC can be analyzed by direct infusion on the Q-Exactive Plus ESI mass spectrometer. Therefore samples should be dissolved in 50% acetonitrile / 0.1% formic acid (v/v) or 50% methanol / 0.1% formic acid (v/v). Alternatively, the molecular weight determination can be performed on an UHPLC-ESI-MS system (Agilent 1290 UHPLC coupled to Q-Exactive Plus ESI mass spectrometer).

Targeted quantification of metabolites by SRM

Targeted quantification of metabolites by SRM (Selected Reaction Monitoring) can be performed on a 5500 QTRAP ESI mass spectrometer (AB SCIEX) that is coupled to a 1290 Infinity UHPLC (Agilent). For SRM experiments the m/z ratio of the target metabolites and the m/z ratios of specific fragment ions of the target molecule must be known. With this information targeted metabolites can be can be detected and quantified with high sensitivity even in very complex mixtures, since the chemical “noise” is relatively low due to the selection of specific ions by the quadrupoles of the QTRAP MS.

To set up a SRM experiment we need to know which metabolites should be quantified, the exact m/z ratios of these metabolites as well as the m/z ratios of characteristic fragment ions of the target metabolites (if already known from literature or own experiments). An absolute quantification is also possible, but requires a calibration curve. For this purpose the target metabolite(s) are needed as standard substance(s). Standard substances of target metabolites are also useful for the optimization of the mass spectrometry parameters of the SRM assay.

Metabolites Profiling/Metabolomics

Mass spectrometry based metabolomics is a non-selective, comprehensive approach for the identification and relative quantification of all (or as many as possible) metabolites in a biological system under different (physiological) conditions. For these experiments a Q-Exactive Plus ESI mass spectrometer coupled to a 1290 Infinity UHPLC is available at the CFH. Mass spectrometry metabolomics experiments produce large data sets which have to be analyzed statistically to identify significant variations that occur at the metabolome level of a biological system. For this purpose suitable software packages (Compound Discoverer 2.0, XCMS online Version v3.5.1) are available.

Due to their different chemical structure (e.g. lipids and monosaccharides) metabolites show different extraction properties and different chromatographic behavior, which makes it difficult to analyze all metabolites of a biological system in a single mass spectrometry analysis. Therefore often approaches are used that focus on defined substance classes (“metabolite profiling”) that can extracted and analyzed together. A holistic approach that covers all metabolites in a biological system (“metabolomics”) is a significant analytical challenge and can only be achieved by using several extraction methods and complementary analytical platforms (GC-MS, LC-MS, NMR). Please arrange an appointment for a consultation to discuss suitable sample preparation and mass spectrometry analysis methods for your metabolomics or metabolite profiling project (see contact).

2D Electrophoresis (2D DIGE)

2D difference gel electrophoresis (2D DIGE) and is an alternative to protein quantification by LC-MS. Compared to conventional 2D gel electrophoresis, 2D DIGE has several advantages: Protein extracts from two different samples are labelled with two different fluorescent dyes (Cy3 and Cy5), pooled, and run on a single 2D gel. The gel is then scanned at the different wavelengths corresponding to the emission maxima of the two dyes to obtain two images, i.e., Cy3 (control) and Cy5 (treated). The two images are overlaid for identification of differentially abundant proteins, which avoids gel to gel variability for dual simple comparisons. A third dye (Cy2) can be used to label an internal standard created by pooling an equal aliquot of all biological samples in an experiment. The internal standard is then run on every gel in the experiment. This allows normalization of the spot volumes and thus more accurate quantification of spots between gels and more accurate analysis of biological variations.

The 2D DIGE technique is highly sensitive and is able to detect proteins in the range of 0.1-1.0 ng with a wide dynamic range up to five orders of magnitude. For a standard minimal labelling experiment (e.g. approximately 3% of the sample is labelled) you will need min. 50 µg of protein extract per sample. An additional advantage of this system is the ability to detect post-translational modifications, such as phosphorylation, glycosylation, etc. that are differentially abundant. Differentially abundant protein spots can be removed from the gel, subjected to MALDI-TOF-TOF or nanoLC-ESI-MS/MS analysis and then identified by a database search (Mascot, Sequest, Andromeda).

The equipment for 2D DIGE experiments is available at the CFH (see equipment). The 2D DIGE experiments have to be carried out by the users themselves. The CFH team will support you in establishing a suitable sample preparation and will assist you in analysis of DIGE-gels with the SameSpots software. The identification of differentially abundant proteins by mass spectrometry is available as service analysis.