Francesco L. Brancia

Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides.

Analysis of tryptic digests of proteins using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry commonly results in superior detection of arginine-containing peptides compared with lysine-containing counterparts. The effect is attributable in part to the greater stability of the arginine-containing peptide ions associated with the sequestration of the single ionizing proton on the arginine side-chain. Reaction of peptides with O-methylisourea resulted in conversion of lysine to homoarginine residues with consequent improved detection during MALDI-MS. Analysis of the underivatized tryptic digest of the yeast protein, enolase, revealed peptides representing 20% of the protein; the corresponding figure after derivatization was 46%.

Towards a truly integrative biology through the functional genomics of yeast

A complete library of mutant Saccharomyces cerevisiae strains, each deleted for a single representative of yeasts 6000 protein-encoding genes, has been constructed. This represents a major biological resource for the study of eukaryotic functional genomics. However, yeast is also being used as a test-bed for the development of functional genomic technologies at all levels of analysis, including the transcriptome, proteome and metabolome.

A combination of chemical derivatisation and improved bioinformatic tools optimises protein identification for proteomics.

The identification of individual protein species within an organisms proteome has been optimised by increasing the information produced from mass spectral analysis through the chemical derivatisation of tryptic peptides and the development of new software tools. Peptide fragments are subjected to two forms of derivatisation. First, lysine residues are converted to homoarginine moieties by guanidination. This procedure has two advantages, first, it usually identifies the C‐terminal amino acid of the tryptic peptide and also greatly increases the total information content of the mass spectrum by improving the signal response of C‐terminal lysine fragments. Second, an Edman‐type phenylthiocarbamoyl (PTC) modification is carried out on the N‐terminal amino acid. The renders the first peptide bond highly susceptible to cleavage during mass spectrometry (MS) analysis and consequently allows the ready identification of the N‐terminal residue. The utility of the procedure has been demonstrated by developing novel bioinformatic tools to exploit the additional mass spectral data in the identification of proteome proteins from the yeast Saccharomyces cerevisiae. With this combination of novel chemistry and bioinformatics, it should be possible to identify unambiguously any yeast protein spot or band from either two‐dimensional or one‐dimensional electropheretograms.

The exploitation of selective cleavage of singly protonated peptide ions adjacent to aspartic acid residues using a quadrupole orthogonal time-of-flight mass spectrometer equipped with a matrix-assisted laser desorption/ionization source

Abstract A series of singly charged tryptic peptide ions were analyzed by tandem mass spectrometry using a quadrupole/time-of-flight instrument equipped with a matrix-assisted laser desorption/ionization source. Highly selective cleavage C-terminal to aspartate, but not glutamate, residues was observed for C-terminal arginine-containing peptides, consistent with earlier findings. Increasing the basicity of C-terminal lysine residues by conversion to homoarginine promoted selective cleavage adjacent to aspartate residues. In contrast, reducing the basicity of C-terminal arginine residues by conversion to dimethylpyrimidylornithine abolished selective backbone cleavage and allowed the formation of multiple sequence ions. The increase in database search selectivity incorporating the single-residue sequence tag information revealed by aspartate-specific cleavage was determined by simulations using the yeast proteome, it was shown that an average of 83% of proteins can be identified on the basis of the mass of a single tryptic peptide together with knowledge of the presence and location of an aspartate residue.

Transcriptome and proteome analysis of osteocytes treated with nitrogen-containing bisphosphonates.

We combined high-throughput screening of differential mRNAs with mass spectrometric characterization of proteins obtained from osteocytes untreated and treated with Risedronate. Microarray analysis revealed, upon treatment, a marked upregulation of messengers encoding zinc-proteins. MS analysis identified 84 proteins in the osteocytes proteome map. Risedronate affected the expression of 10 proteins, associated with cytoskeleton, stress-response and metabolism. Data validated using gel imaging in combination with the GLaD post digestion isotopic labeling method provide the molecular basis for understanding the role of bisphosphonates as antiapoptotic drugs for osteocytes.

Mapping the stability diagram of a digital ion trap (DIT) mass spectrometer varying the duty cycle of the trapping rectangular waveform

In a digital ion trap the βr and βz boundary lines of the stability diagram are determined experimentally using an innovative approach. In the rectangular waveform-driven digital ion trap (DIT) manipulation of the waveform duty cycle allows introduction of a precisely defined DC quadrupole component into the main trapping field. Variation of the duty cycle can be controlled at software level without any hardware modification. The data generated use peptide ions, which produce stability diagrams in good agreement with the theoretical stability diagrams previously determined by simulation studies.

Bioinformatic assessment of mass spectrometric chemical derivatisation techniques for proteome database searching.

Identification of proteins from the mass spectra of peptide fragments generated by proteolytic cleavage using database searching has become one of the most powerful techniques in proteome science, capable of rapid and efficient protein identification. Using computer simulation, we have studied how the application of chemical derivatisation techniques may improve the efficiency of protein identification from mass spectrometric data. These approaches enhance ion yield and lead to the promotion of specific ions and fragments, yielding additional database search information. The impact of three alternative techniques has been assessed by searching representative proteome databases for both single proteins and simple protein mixtures. For example, by reliably promoting fragmentation of singly‐charged peptide ions at aspartic acid residues after homoarginine derivatisation, 82% of yeast proteins can be unambiguously identified from a single typical peptide‐mass datum, with a measured mass accuracy of 50 ppm, by using the associated secondary ion data. The extra search information also provides a means to confidently identify proteins in protein mixtures where only limited data are available. Furthermore, the inclusion of limited sequence information for the peptides can compensate and exceed the search efficiency available via high accuracy searches of around 5 ppm, suggesting that this is a potentially useful approach for simple protein mixtures routinely obtained from two‐dimensional gels.

Digital ion trap mass spectrometer for probing the structure of biological macromolecules by gas phase X-ray scattering.

Small-angle X-ray scattering is a technique for the characterization and structural analysis of a variety of materials including biological macromolecules and polymers. For the conformational analysis of proteins, the interaction between sample and X-rays is generally performed when the proteins are present in solution. Here a three-dimensional digital ion trap interfaced with a high intensity X-ray source is built to prove that X-ray scattering can be performed on ions isolated in gas-phase. Initial experiments on an unresolved ion population of multiply charged cytochrome C ions indicate that a small-angle X-ray scattering signal can be detected and that partial structural information can be extracted about the overall molecular structure of protein ions.

A density functional theory (DFT) study on gas-phase proton transfer reactions of derivatized and underivatized peptide ions generated by matrix-assisted laser desorption ionization

In this study, classic molecular dynamics (MD) simulations followed by density functional theory (DFT) calculations are employed to calculate the proton transfer reaction enthalpy shifts for native and derivatized peptide ions in the MALDI plume. First, absolute protonation and deprotonation enthalpies are calculated for native peptides (RPPGF and AFLDASR), the corresponding hexyl esters and three common matrices α-cyano-4-hydroxycinnamic acid (4HCCA), 2,5-dihydroxybenzoic acid (DHB), and 6 aza-2-thiothymine (ATT). From the proton exchange reaction calculations, protonation and deprotonation of the neutral peptides are thermodynamically favorable in the gas phase as long as the corresponding protonated/deprotonated matrix ions are present in the plume. Moreover, the gain in proton affinity shown by the ester ions suggests that the increase in ion yield is likely to be related to an easier proton transfer from the matrix to the peptide.

Offline and Online Liquid Chromatography Mass Spectrometry in Quantitative Proteomics

Hyphenation with liquid chromatography has become indispensable in mass spectrometry-based proteomics. Sample complexity together with the large variations in dynamic range can be only tackled using techniques that isolate and/or concentrate individual components prior to mass spectrometric analysis. In this review the most recent developments in micro/nanoliquid chromatography interfaced with MALDI and electrospray ionisation are discussed. Particular attention is focused on all applications related to quantitative proteomics.