Biopolymers & Proteomics Laboratory

The David H.Koch Institute for Integrative Cancer Research at MIT

Bldg 76 Room 181

Telephone: 617-253-7038

MALDI-TOF MS

Overview of the Technology

We are using the Applied Biosystems Model Voyager DE-STR instrument for Matrix Assisted Laser Desorption Instrument Time of Flight (MALDI-TOF) mass spectrometry. MALDI-TOF mass spectrometry works on the principle that ions of different molecular weights travel at different speeds, providing they are accelerated with the same potential and carry the same charge. Fortunately, most MALDI ions are singularly charged (MH+) i.e. carry one extra proton (+H) or have been singularly deprotonated. With larger molecules, you may see a small amount of the MH++ and perhaps MH+++ ions. The time of flight is proportional to mass--heavier ions take longer to reach the detector. For a more thorough explanation, please see chapter 1.3 of the manufacturer's operators manual entitled "Voyager Biospectrometry Workstation User's Guide" which can be downloaded from the Applied Biosystems web page or from our Biopolymers FTP server.

Basically, the sample is mixed with matrix solution to assist ionization and then dried on a sample plate. A laser beam is fired at the sample crystals simultaneously with the application of a high voltage pulse. The time it takes for sample ions to drift through the flight tube to the detector is proportional to their molecular weight.

The fact that the sample is initially in crystal form reduces the ability to get information on the quantity of sample loaded. The analysis of one well-formed crystal in a very dilute sample can give a greater signal than the data from the analysis of multiple, poor-quality crystals in a far more concentrated sample. If you need quantitative data, you are better off using an ESI mass spectrometer, where sample is dissolved and sprayed into the MS. The amount of sample is directly proportional to the signal for that sample.

The MALDI mass detection range for peptides/proteins is between 500 and 350,000 Da. Ions below 500 Da can be measured if the amount of sample (usually ca. 10+ pmole) provides a signal that sufficiently exceeds those of the numerous matrix artifact ions. In general, we prefer to analyze small molecules on our ESI instrument, which provides valid data in the 30 to 80,000 Da range.

Sample Requirements

  • General Protein and DNA Samples
    • We would ideally like to load 1-100 pmol of peptide, 10-100 pmol of protein, and 10-100 pmol of DNA. As for volume, we generally mix 1 ul of your sample with 1 ul of matrix. We will concentrate the sample if necessary.The sample should be free of salts and detergents because these may inhibit ionization. We can desalt your sample using reverse phase or ion exchange resin. Removal of detergent is often sample-specific, so we prefer that you do this yourself. You should precipitate or dialyze your sample away from detergent or contact us about other alternatives (also see ESI Sample Requirements). Since MALDI analysis is inexpensive, you may want to rely on the dilution of your sample to negate the effects of detergent and salt, and if that fails, proceed with sample clean-up. Remember, 1 ul of a 1 pmol/ul solution is more than sufficient in most cases.
  • Protein-DNA Conjugates
    • Tell us the MW of each portion, as this affects how we go about the analysis.
  • Mass Mapping for Protein Identification Samples
    • We would like 1 pmol of in-gel protein, but as little as 125 fmol is potentially feasible from CBB (e.g. G250) or silver stained gels (a special silver stain protocol is required--see below). Consider newer fluorescent stains such as Sypro Ruby from Molecular Probes in lieu of silver stain. Sypro ruby appears to be give better mass spectrometry results and is easier to use. We appreciate a control gel slice from a blank area of the gel of approximately equal size to the sample to enable subtraction of gel artifact ions prior to database searching.Do the required de-stain procedure, excise the band of interest, and place in an eppendorf tube. Keep the protein/gel ratio as high as possible, i.e. trim off any non-protein containing gel. Keep frozen, if possible.

MALDI Mass Fingerprint Mapping for Protein Identification

Most of the samples we process for MALDI mass fingerprint mapping for protein identification come from SDS-PAGE bands that have been stained with either CBB or silver. We can also assist in HPLC of protein intended for digestion and mapping. Solution phase digestion provides better coverage. We perform a tryptic digest on the gel band or sample, isolate the resulting peptides, and measure their ion molecular weights at high accuracy. We generate a list of ions that are present in the sample but absent from the control band and do a database search to determine the identity of the sample. It is most helpful if the number of proteins in the band are minimized. When more than two or three proteins are present the search becomes difficult. Use a 2D gel if necessary to reduce the sample complexity. We would much rather have 200 fmol of a single protein than 1 pmol of a mixture. Use our Ion Trap protein identification service for identifying gel bands that are likely to contain more than one protein or to identify the proteins in an immunoprecipitation prior to SDS-PAGE separation.

We have found that CBB stained samples of 250 fmol (we are talking about sample in the gel band, not sample loaded onto the gel) or greater work virtually 100% of the time, while CBB samples of 125 fmol work about 50% of the time. One must be especially careful with silver stained samples to avoid keratin contamination, which can dwarf the signal from the actual sample.

If using silver stain, please ask for our hand-out notes regarding keratin contamination elimination and reagent changes to make the stained band more compatible with trypsin digestion. Also see: Gharahdaghi, F., Weinberg, C., Meagher, D., Imai, B., and Mische, S. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity. Electrophoresis 20, 601-605 (1999).back to top

Data Output

Data will be emailed in a Word document. The data file can be saved as a text file which can be opened in Excel and plotted in histogram format to give a reasonable graphic. Unfortunately, our instrument spectra files can only be opened using the manufacturer's software. You are welcome to use this software on our instrument computer to expand small segments of the spectra and examine them in detail.

  • General Protein or DNA Output
    • We usually provide at least two printouts of the spectra taken. One will show the entire range of the spectrum measured and some will detail the masses of interest. We often provide you with a spreadsheet file that details the intensity (peak height) and peak area for each mass measured.
  • Mass Mapping for Protein Identification Output
    • We provide you with enough of the output from a database search program, usually Protein Prospector from UCSF (MS-FIT), that we think is necessary to identify the protein(s) present in the sample. To see if more than one protein is present, we subtract the ions used to identify the first protein and do a second search.This is done repeatedly until no further matches are discerned.
      The MOWSE score for the actual protein present is usually several orders of magnitude higher than the score of other proteins listed in the output report. The grid display is very useful in visualizing which protein identifications were returned from which ions.

      Most importantly, we provide a manually generated peak list for all ions found that were not present in the control gel band. You can cut and paste this list into Protein Prospector or any other search program and vary the parameters to see if different results are obtained. You can use the peak list to re-search at a later date when the genome of interest is more complete. We "de-isotope" the peak list for you, i.e. we report only the C12 isotope for each ion.

      We use internal calibration, unless otherwise noted, and this results in less than 10 ppm error, with the majority of ions having less than 5 ppm error. Good precision (a consistent error bias) is indicative of a high confidence match, e.g. most of the matching ions are all of negative 3 ppm error +/- 2 ppm. On the other hand, if a matching peptide had to be modified by two oxidized methionines, three phosphates, and a pyroglutamic acid conversion in order to align with an ion from the peak list, and the MW error is positive 25 ppm, while all the other ion errors had an error of negative 3 ppm +/- 2 ppm...it is unlikely that that particular peptide match is valid.

      If you requested to do the database search portion of the process yourself, we provide you with a spreadsheet file that details the intensity (peak height) and peak area for each mass measured. The peak list and de-isotoping are generated automatically by the instrument software and are not verified. Any trypsin and gel blank artifact peaks are not subtracted.

Click here for an explanation of the code that are included in your MALDI data.

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