Suzana Martinović

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by “mass locking” with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations.

Proteome analysis using selective incorporation of isotopically labeled amino acids

A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content. An initial demonstration was conducted for proteins isolated from Escherichia coli (E. coli) using a multiple auxotrophic strain of K12. Proteins extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum. The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein. Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein. Preliminary results show the efficacy of this method for unambiguously identifying proteins isolated from E. coli.

Mass spectrometic detection for capillary isoelectric focusing separations of complex protein mixtures

Capillary isoelectric focusing (CIEF) can provide high‐resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection. This technique would be greatly enhanced by much more information‐rich detection methods that can aid in protein characterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization. Studies have revealed 400—1000 putative proteins in the mass range of 2—100 kDa from total injections of ˜ 300 ng protein in single CIEF‐FTICR analyses of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for quantitative proteome‐wide measurements of protein expression. The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes.

Creating nanocavities of tunable sizes: Hollow helices

A general strategy for creating nanocavities with tunable sizes based on the folding of unnatural oligomers is presented. The backbones of these oligomers are rigidified by localized, three-center intramolecular hydrogen bonds, which lead to well-defined hollow helical conformations. Changing the curvature of the oligomer backbone leads to the adjustment of the interior cavity size. Helices with interior cavities of 10 Å to >30 Å across, the largest thus far formed by the folding of unnatural foldamers, are generated. Cavities of these sizes are usually seen at the tertiary and quaternary structural levels of proteins. The ability to tune molecular dimensions without altering the underlying topology is seen in few natural and unnatural foldamer systems.

Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing — mass spectrometry

The human liver alcohol dehydrogenase (ADH) isoenzymes are currently believed to play a major role in ethanol metabolism, accounting for most of the ethanol oxidized in the liver. They have similar molecular masses and similar isoelectric point (pI) values (the 13 possible isoenzymes having pIs in the range of 8.26—8.87), making their characterization a significant analytical challenge. Capillary isoelectric focusing (CIEF) coupled on‐line with electrospray ionization — Fourier transform ion cyclotron resonance (ESI‐FTICR) mass spectrometry was applied to separate and characterize mixtures of αα, β1β1 and β3β3 ADH isoenzymes. Seven different species were resolved by the separation in the pI 8.26—8.67 range. ESI‐FTICR analysis of native ADHs revealed that each noncovalent ADH complex contains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well‐suited for detailed characterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.

Capillary isoelectric focusing--mass spectrometry of proteins and protein complexes.

Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole-cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of noncovalent complexes on the intact complex level.