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Biomolecular Mass Spectrometry

We have two mass spectrometer systems, both from Applied Biosystems. The first is a Voyager DE Pro. It is a Matrix-Assisted Laser Desorption Ionization time-of-flight mass spectrometer (MALDI-TOF). Samples may be run in either positive- or negative-ion mode. Samples are ionized utilizing delayed extraction, which results in high resolution. Molecules up to about 5000 Daltons are analyzed in reflector mode for enhanced resolution, while larger molecules are analyzed in linear mode.

The second instrument is a QSTAR Elite hybrid quadrupole-Tof mass spectrometer.
This mass spectrometer is equipped with both a nanospray and electrospray ionization sources which can be used to produce either positive or negative ions. Samples are generally dissolved at concentrations of 0.2 to 0.5 micrograms/microliter in a solution of a dilute (0.1-1.0% v/v) volatile organic acid in water. The acids commonly used are acetic, formic or trifluoroacetic acid. Often an organic solvent such as methanol or acetonitrile is added to increase the sensitivity and the degree of ionization. The presence of detergents will usually completely suppress ionization, and thus prevent the collection of any useful data!

Precise molecular weights can be determined for proteins (including glycoproteins) and peptides by mass spectrometry. The measurement is accurate down to +/- 0.01%, or +/- 5 Daltons for a 50 kDa protein. As little as 20 to 40 picomoles of protein (1.0 to 2.0 micrograms for a 50 kDa protein) or 20-40 femtomoles of peptide is sufficient for analysis if optimal conditions are used. Proteins up to 100 kDa in mass have been successfully analyzed by this type of mass spectrometer system. Mass spectrometry is particularly powerful for the identification of post-translational modifications. The specific location of the modifications can be determined by on-line HPLC/mass spectrometry of a proteolytic digestion mixture of the protein. Eluted peptides can be identified by comparison of their observed masses with the calculated masses of the predicted proteolytic peptides, if the protein sequence is known. Any observed peptide masses with unmatched calculated masses are candidates for the post-translationally modified peptides.

Samples may be introduced into the mass spectrometer by (1) capillary, (2) direct syringe pump infusion or by (3) on-line capillary or nano HPLC. The HPLC method is slower, but it provides a means of desalting and it can be used to better analyze complex mixtures.

Mass spectrometry of peptides is generally easy, proteins are less predictable. Unfortunately, not all proteins will produce data. The larger the molecule the more difficult. Small molecules (<20000 daltons) are easy and a wide range of solvent and instrument parameters will result in good data, while larger proteins are more difficult. Larger proteins only yield data in a very narrow range of conditions, and those conditions are determined mostly by trial and error. The degree of difficulty is related to factors such as protein solubility at acidic pH, the protein's isoelectric point, and the number of disulfide bonds.

LC/MS/MS

Samples containing up to twelve proteins in mixture may be identified by performing a tryptic digestion followed by reversed-phase LC/MS/MS analysis. We utilized a Dionex Ultimate 3000 nanoscale HPLC system connected to Applied Biosystems QSTAR Elite mass spectrometer. Tryptic peptides are separated on a C18 column consisting of 3-micron particles packed in a column of 75 microns ID x 150 mm length. Peptides eluting from the column are surveyed by the first stage of the mass spectrometer to determine their usefulness for MS fragmentation. Peptides meeting the predetermined criteria are subjected to automated MS fragmentation. For each survey scan, up to three peptides are subjected to fragmentation. Finally all collected LC/MS/MS data are submitted to a MASCOT search of an in-house NCBI database to identify proteins present in the original sample. The sensitivity of this method is limited to approximately 50-100 femtomoles of a given protein for a quality identification.

LC/LC/MS/MS (MUDPIT)

Samples containing hundreds of proteins may be subjected to this method for identification. The first dimension is cation exchange chromatography (SCX). Typically from eight to 25 fractions are collected. Each fraction is then run as a separated sample on a reversed-phase column as described above for LC/MS/MS. All LC/MS/MS data from each sample are merged into a single file and submitted to a MASCOT search of an in-house NCBI database to identify proteins present in the original sample. Similar to the LC/MS/MS experiment, the sensitivity of this method is limited to approximately 50 to 100 femtomoles of a given protein for a quality identification. The charge for the analysis is dependent upon the number of first-dimension fractions selected for second-dimension LC/MS/MS. A total charge of $855 includes a first-dimension SCX separation and fraction collection followed by second-dimension LC/MS/MS of eight of the fractions. This includes the MASCOT database search. The MASCOT data is provided to the client as a website link. The client can then view as much or as little of the supporting data as desired.

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• Mass Spectrometry order form (requires acrobat reader)
  

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