created: 14th January 1998, last updated: 14th January 1998, © 1998 ABRF
A.L. Burlingame organized the ABRF minisymposium entitled "Mass Spectrometry" at the Technology Corners session. Burlingame, R.T. Aplin and K.R. Clausser spoke on various uses of mass spectroscopy. Note that complete abstracts appear in the FASEB Journal Volume 11, No. 9, July, 1997 if you want additional details.
A.L. Burlingame, UCSF, "Tools for studying the proteome in the context of human disease".
A.L. Burlingame began with the point that in the last twenty years we have seen a progression of the routine study of proteins with the aid of Edman degradation to the routine study of proteins with mass spectrometry (MS). In the field of molecular physiology, studying the function of cells requires knowledge of the genome and the proteome of the cell. Burlingame defined the proteome as encompassing knowledge of the life of every protein in the cell from its synthesis through its degradation. Using MS, proteins can be studied with as little as 1 picomole of material. Collision-induced dissociation (CID) mass spectra currently can be obtained for proteins and their fragments for masses up to 3000 Da. The field is looking to the future where 1 femtomole or 1 attomole will yield mass data. But, to get to this level of sensitivity, what are the important parameters? Sensitivity, mass resolution, mass range, low peptide bond cleavages, selectivity in ionization such as "suppression effects", etc.
The practical points gleaned from Burlingame's talk: Use electrospray MS when determining masses of peptide fragments from tryptic digests as, apparently, MALDI preferentially picks out the arginine-containing peptides. Sample cleanup is required for electrospray analysis; particularly samples containing nonvolatile salts and detergents.
R.T. Aplin, Oxford University, "Electrospray ionization mass spectrometry: a probe for the study of enzyme mechanisms".
Aplin uses electrospray MS to study cell-free enzyme kinetics. Several examples were given including determination of the rate of oxidation of penicillin , a b-lactam, with b-lactamase; the biosynthesis of b-lactam antibiotics; the kinetics of porcine pancreatic elastase; and clavulanic acid biosynthesis.
Aplin uses the mass spectrometer to observe reaction intermediates, products, complexes, etc. Using deuterated water for the solvent, Alpin follows reactions that involve the addition of a proton from the solvent. Where there is ester bond formation, he does the experiment in an O18 atmosphere to see the addition of the oxygen. Where there are complexes formed (such as the 1:1 complexes of elastase and inhibitors) he follows the time course of the conversion from the complex back to the free enzyme. In clavulanic acid biosynthesis, where there is a loss of a carboxyl group (-44), he follows the addition of an alkali salt (K) with potassiation of the acidic residues (+38).
C.R. Jimenez, K.W. Li, S.J. Fisher, and A.L. Burlingame, UCSF, "MALDI tandem mass spectrometry for the direct qualitative and semi-quantitative analysis of neuropeptides in single cells and tissue".
As a biologist who interacts with several neuroscientists, I was impressed with Connie Jimenez's talk on the use of MALDI to study neural peptides. Her interest was to follow the processing of the peptides in single neurons in the brain of the pond snail. Mass information is used to monitor peptides that have already been identified, to recognize consensus sites for processing of prohormones, and to predict posttranslational modifications. MALDI is particularly useful due to its high sensitivity, ability to look at mixtures, and tolerance of salts. Single cells are ruptured with the dihydroxybenzoic acid matrix. The matrix also conveniently inhibits proteases that are present. Sequence data from peptides is further obtained by tandem mass spectroscopy. A phosphorylation site was identified in a peptide (+80) based on an observed mass change.
K.R. Clauser, R.A. Foulk, P.R. Baker, S.J. Fisher, and A.L. Burlingame, USCF, "Mining genome databases via protein mass spectrometry to understand the pregnancy disorder pre-eclampsia".
Karl Clausser presented a study comparing proteins released by normal chorionic villi in a normal oxygen environment versus a reduced oxygen environment. This was an excellent example of the use of 2-D PAGE to examine the abundance or absence of different proteins in these two systems. Proteins were identified by in-gel tryptic digestion, coupled with mass fingerprinting of the digest by MALDI-TOF, and MS/MS spectra of individual peptides in the digest. Clausser made the point that recent advances in automation of MS instrumentation as well as mass accuracy (1-2 daltons) and sensitivity make possible the identification of large groups of proteins (by searching the databases), such as these obtained from the villi. When looking at unseparated tryptic digests, products from autoproteolysis of trypsin-or even common keratin contaminants-can be used as internal calibrants to increase mass accuracy.
A good web site for identification of proteins from the mass fingerprint data is UCSF's ProteinProspector site (http://prospector.ucsf.edu/). This site includes a number of programs including MS-Fit for protein identification from mass fingerprint data and MS-Edman which even allows for identification of the protein knowing only the molecular weight of a tryptic fragment and some of the amino acid composition. For example, MS-Edman will let you search for a fragment with a mass of 1601.2 daltons and at least 1 methionine, 2 alanines, and 1 aspartic acid.
The presentations were followed by a workshop "Mass Spectroscopy for Biochemists" consisting of an open panel session with the symposium speakers and the audience. The following bits of information were gleaned from the open discussion.
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