Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 3, 2000, pp. 194

Biotecnología Aplicada, Volume 17, July-September 2000, p. 194

Mass Spectrometry in Protein Studies from Genome to Function

P Roepstorff

Department of Molecular Biology, Odense University, DK 5230 Odense M, Denmark.

From a selection of papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999.

Code Number: BA00058

The triumphal progress of DNA sequencing

Advanced technology for determination of DNA sequences has become widely available in the last decade and has been used for sequencing cDNA and entire genomes from a variety of organisms ranging from viruses to human. As a consequence, the amount of DNA sequence information entered in publicly accessible databases has increased exponentially in the last decade. This growth has by far exceeded the growth of sequences entered in databases based on protein sequencing. In addition to the genomic sequencing, large-scale partial cDNA sequencing has resulted in another set of data, the so-called expressed sequence tags containing stretches of sequence from a large number of genes from a variety of organisms. The genomic sequences, however, only provide information about the potencial of the selected microorganisms and cell types but do not reflect the actual situation at any given moment, i.e. which proteins are expressed and how they are modified. cDNA sequences or the incomplete ESTs gives information on the actually expressed proteins, but no information on processing and secondary information. Therefore, studies of the proteins will never be obsolete.

Mass spectrometry has conquered protein analysis

Independently, but coinciding in time, mass spectrometric analysis has undergone an equally dramatic development. From being an analytical tool for analysis of small volatile molecules, new ionization methods, especially electrospray ionization (ESI) [1] and matrix assisted laser desorption/ionization (MALDI) [2], have increased the accessible mass range to include nearly all proteins. Mass accuracy and sensitivity have been improved to routinely allow molecular mass determination on the 100 ppm level or better of peptides and proteins available in only mid to low femtomole amounts [3]. Mass spectrometry (MS) has been proven ideal for analysis of peptide and protein mixtures and partial or complete sequence information can be generated from the single components in such mixtures by the so-called MS/MS techniques. In addition, MS is the ideal technique for analysis of post translational modifications in proteins [4], thus being the perfect complement to DNA sequencing.

Proteome analysis: the next step after the genome

Once a genome is sequenced, then the next natural step is analysis of the proteome, which as defined by Wilkins et al. [5] represent: "The total protein complement expressed by an organism, a cell or a tissue type". Proteome analysis involves two essential steps. Firstly, separation and visualization of the proteins and, secondly, identification of the proteins relative to the genomic sequence, if known. 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is presently the only technique for separation of all or the majority of the proteins from a given cell type. Identification of the proteins is now routinely carried out by mass spectrometry after proteolytic digestion of the proteins in the gel. This can be done either based on peptide maps produced by MALDI MS or partial sequences produced by ESI MS (For a complete strategy see: Shevchenko et al. [6], Jensen et al. [3]. Of these techniques peptide mapping by MALDI MS is the simplest and most sensitive, whereas the sequence based techniques are more specific. Partial sequences also often allow identification in cases where only partial protein sequence information is available, e.g., in EST databases [7]. Upon positive identification, the corresponding cDNA can then be ordered and sequenced.

Characterization of secondary modifications is essential

Once a protein is identified, then the next obvious questions are: Are the identified proteins post translationally modified and if so, how? If the purified protein is available, then the strategy is to compare its molecular mass determined by MS with that calculated based on the DNA sequence. If these masses are different, then the modified sites and the types of modification are identified based on mass spectrometric peptide mapping if relevant supplemented with MS/MS of relevant peptide ions or degradation with appropriate enzymes, e.g., glycosidases or phosphatases [8, 9]. If the proteins are only available as spots or bands in electrophoretic gels, then it is often not possible to determine the molecular mass of the intact protein, and characterization of post translational modifications must rely on peptide mapping before and after enzymatic treatments and when appropriate MS/MS. In such cases it is essential to obtain complete or very high sequence coverage in the peptide maps [10, 11].

Studies of protein interaction and higher-order structures

The acceptance of mass spectrometry as a tool for studies of protein interaction and protein higher-order structures is gradually increasing [4]. Interaction screening based on "affinity fishing" followed by mass spectrometric identification of the bound proteins shows great promise [12]. Studies of protein conformation by deuterium exchange, of surface exposed residues by specific labeling or limited proteolysis, and of the interaction interface by cross-linking, although not competitive with techniques like X ray crystallography and NMR spectrometry in terms of amount of detailed information, can yield valuable information on much smaller protein quantities.

Conclusion

Mass spectrometry is now a viable tool on all levels in studies of proteins from genome to function.

References

Copyright Elfos Scientiae 2000