created: 14th January 1998, last updated: 14th January 1998, © 1998 ABRF
The minisymposium on Protein Chemistry, organized by Kenneth Walsh, generated so much interest among meeting attendees that organizers were forced to move it to the largest auditorium in the Moscone Convention Center. Because the most remarkable achievements in protein chemistry in the last several years are associated with protein mass spectrometry, not surprisingly, all talks described applications of MS for protein identification and characterization.
Kenneth Walsh opened the symposium and introduced the speakers. In his brief presentation, he spoke about the "Current Challenges in Protein Chemistry". The major technological advances in DNA sequencing resulted in a rapid accumulation of protein sequence data. This has freed protein chemists to focus on methods that identify trace quantities of specific proteins with their DNA counterparts, and upon procedures that identify posttranslational modifications. Consequently, current goals of protein chemistry are: 1) protein identification, i.e., relate proteome to genome; 2) characterization of proteins (e.g., recombinant proteins, posttranslational modifications, etc.).
Virtually all eukaryotic proteins appear to be posttranslationally modified by covalent additions or deletions during their biosynthesis or cellular regulation. Although some posttranslational modifications (PTM) can be predicted from motifs observed in DNA sequences, the most generally applicable technique for their identification, nowadays, is protein mass spectrometry. The first step in PTM detection is comparison of the observed protein mass with that predicted from the cDNA sequence followed by examination of a peptide digest. Modern methods of mass spectrometric analysis of peptides (ESI-MS and MALDI-TOF) offer advantages of speed and sensitivity compared to traditional methods of protein identification based on microapplication of the Edman degradation.
Ruedi Aebersold, University of Washington, "Identification and characterization by ESI-MS/MS of proteins separated by gel electrophoresis."
Aebersold's presentation described technology developed for the establishment of protein expression profiles in cells and for the identification and localization of regulatory modifications within the polypeptide chain. Critical issues in the development of the technology were: i) increasing sensitivity; ii) manipulating small samples; iii) removing contaminants; and iv) minimizing the need for HPLC separation. Accordingly, the separation of proteins was achieved on 2-D gel electrophoresis followed by capillary chromatography or capillary electrophoresis with tandem mass spectrometry. The concentration limit of detection was improved by on-line coupling of a solid-phase microextraction device to a capillary electrophoresis-tandem mass spectrometry system. The analyte proteins are identified by searching sequence databases with the information contained in the collision-induced spectra of selected peptides. The sensitivity achieved with this system was at the low attomole per microliter level.
Steven Carr, SmithKline & Beecham, "Posttranslational modifications of proteins."
Nearly 200 structurally distinct covalent modifications have been identified thus far, ranging in size and complexity from conversion of amides to carboxylic acids, to the attachment of multiple complex oligosaccharides. Several factors such as protein structure and sequence, host cell, or bioprocess environment can effect PTM. Mass spectrometry is uniquely suited for establishing the presence, location, and chemical nature of blocking groups and other PTM. A two-dimensional phosphopeptide mapping approach was developed in the author's laboratory. In the first dimension, LC/MS is used to analyze an enzymatic digest of the phosphoprotein. The instrument is optimized to produce and detect CID-generated product ions. HPLC fractions containing phosphorylated peptides are analyzed in the second dimension. The second dimension utilizes nanoelectrospray MS to perform precursor ion scans on targeted fractions. The precursor ion scan produces molecular ions only for those peptides that can produce a phosphate specific product ion. Therefore, the molecular weight of the modified peptide can be determined in this dimension even in the presence of other peptides. The modified peptides can then be sequenced from the same sample loading by acquiring a full scan product ion spectrum.
Two speakers presented at the associated workshop; Len Packman and A.L. Burlingame:
Len Packman, Cambridge University, "Spots before your eyes -- identifying proteins in 2-D gels by MALDI-TOF MS and composition analysis."
Packman described a strategy for identifying proteins in gels that can be performed in the average biochemistry laboratory. The first step is separation of the protein mixture using SDS-PAGE. Further manipulation can be performed either with gel-entrapped protein or a similar protein transferred to a membrane. In the next step, the protein spot is cut from the gel or membrane and subjected to analysis. The analysis can consist of amino-terminal sequencing, amino acid composition analysis and the mapping of the masses of proteolytically generated fragments. The latter analysis can be done by MALDI-TOF MS on relatively low cost instrumentation. The data obtained are submitted to one of the existing databases (e.g., MOWSE, MS-Fit). Because many proteins already exist in the database, techniques aimed at comparing characteristics of the isolated proteins with similar characteristics of proteins in the database can lead to a match and positive identification. The lack of a match might indicate an unknown protein.
A.L. Burlingame, UCSF, "Identification of Gel-Separated Proteins by Mass Spectrometric Partial Peptide Sequencing and Database Interrogation."
This presentation centered around the application of mass spectrometry in protein chemistry. Currently, electrospray ionization using microbore or capillary HPLC at nanoliter/minute sample flow rates is a powerful tool for rapid protein identification of 2-D gel-separated proteins using database interrogation strategies. This approach may be coupled with orthogonal time-of-flight or quadrupole analyzers, and provides more complete analysis of a digest when de novo sequence information or the characterization of a protein is desired. Tandem methods provide unambiguous sequence information for design of oligonucleotide probes and for structural investigations.
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