FAQ

1. Which informations about the sample should be provided?

Information about structure, sequence, chemical reactivity, pH-value, contaminations, solvents, etc. simplify the processing of the samples and accelerate the MS analysis. The more detailed the information about the sample is, the better! Please include the available information in our sample submission sheet and submit it together with your samples. For an identification of proteins from SDS-gels, please attach an image of the gel on which bands or spots of interest are marked.


2. Which kind of molecules can be analysed by MALDI-TOF?

In a MALDI-TOF measurement sample molecules are ionized from cocrystals of matrix and sample molecules by irradiation with a laser beam. In principle all molecules are detectable, which can be ionized this way. There are restrictions concerning the molecular weight of the sample molecules: for substances with a molecular weight ≤ 600 Da matrix signals might interfere with or suppress the sample ions.


3. Which sample concentration is required for a mass determination by MALDI-TOF?

The concentration for a MALDI-TOF analysis of proteins should be in the range of 10-100 pmol/µl, with a minimum volume of 5µl. For peptides, oligosaccarides and oligonucleotides 1-50 pmol/µl are sufficient, again in a volume of at least 5 µl. Modified proteins and peptides (e.g. phosphorylation, glycosylation, etc.) are frequently ionized with less efficiency, therefore the concentrations of this substances should not be at the lower limit of the ranges denoted above. If you are submitting lyophilized samples for analysis, please enter the dry weight of the sample on the sample submission sheet.


4. Which kind of buffers should I use for my sample preparation?

When you are preparing your samples, please try to avoid buffers with ionic detergents like SDS, CHAPS or non-volatile solvents like DMSO. Also critical are glycerol, Tween20, TritonX100 or PEG, because these substances interfere with the cocrystallisation of sample and matrix. Additionally these substances are easily ionized themselves and produce intense ion signals, which may completely suppress the signals of the sample molecules. If these chemicals are absolutely necessary for the sample preparation, they have to be removed before MS analysis by suitable purification steps (RP-HPLC, SPE, etc.)! Salts and buffer substances like sodium chloride, phosphates, Tris or urea are less critical, but lead to adducts with the sample molecules which gives rise to additional signals in the MS spectrum. These substances have to be removed also before the measurement. If possible, use volatile buffer substances like ammonium bicarbonate, ammonium acetate or ammonium formiate. Suitable solvents for the sample preparation are water, acetonitrile or methanol.


5. Which gel staining methods are compatible with mass spectrometry (MS) analysis?

For identification of proteins from SDS-gels, corresponding bands/spots are cut from a 1D- or 2D-gel and digested in gel by a protease (usually trypsin). The resulting peptides are extracted from the gel and analysed by MALDI-TOF or ESI-MS. Successful identification of the proteins is at least partially dependent on the applied gel staining method. The following staining methods are compatible with a subsequent MS analyis:

  1. Conventional Coomassie staining:
    Protein bands, which can be detected by Coomassie staining, are suitable for MS analysis. Usually coomassie-stained proteins can be identified with a peptide mass fingerprint without any problems if they are registered in a protein database.
  2. Colloidal Coomassie Staining:
    The sensitivity of some colloidal coomassie stainings is almost close to the sensitivity that you can reach with silver stainings (detection limit ca. 15 ng). Gels stained with colloidal Coomassie can be used without any problems for subsequent MS analysis. Therefore this staining method is absolutely preferable to silver staining. You can find a protocol for a sensitive colloidal coomassie staining here.
  3. Silver staining:
    The MS analysis of proteins from silver stained gels is possible, if you avoid cross-linking of your proteins with the gel (polyacrylamide) matrix by glutaraldehyde. You can find a suitable protocol here. Alternatively you can use commercial kits for silver staining. The sensitivity of the silver staining is higher compared to coomassie stainings (down to 1 ng protein if you use glutaraldehyde or 3-5 ng protein with MS compatible staining protocols). However, silver stains have a limited linear range, meaning that the actual protein concentration present cannot be estimated correctly through the intensity of the corresponding band or spot. Therefore the amount of protein may not be sufficient for MS analysis.
  4. Fluorescence staining:
    The sensitivity of some commercial fluorescence stains (e.g. Sypro Ruby or Deep Purple), is equal or even higher than silver staining. In this case proteins are detected via imaging systems like the Typhoon-Imager. Besides sensitivity, the advantages of these stains compared to silver staining are a significantly higher linear detection range (important for quantifications) and a better compatibility with MS analysis. A disadvantage of this method is the fact, that the bands or spots can not be cut straight from the gel and a print-out of an image generated by the Imager has to be used as an orientation instead. Technically this problem can be solved by a spotpicker, which cuts the spots from the gel by means of the Imager picture. In contrast to the so-called post-stains it is also possible to label proteins directly with fluorescent dyes before electrophoresis. For 2D DIGE analysis the fluorescent dyes are attached covalently to amino groups (minimal labelling) or cysteins (saturation labelling) of the proteins. Like in the case of fluorescent post-stains, the proteins are detected by an imaging system. The sensitivity is higher compared to post-stains: 0.1-0.2 ng protein (minimal labelling) or 0.005-0.01 ng protein (saturation labelling).



6. Which data will I receive from the Core facility after a successful measurement?

The data (MS-spectra or MS/MS-spectra) will normally be submitted electronically as PDF files. For protein identification via peptide mass fingerprinting or nanoLC-ESI-MS/MS the result of the database search (Mascot, Sequest) is also included.


7.  Is it possible to obtain mass spectrometry data for publications or presentations?

In addition to the pdf-file the data of the spectra can be provided as an Excel file and the spectrum as bitmap or powerpoint file on request.