Biomedical subjects

Daniel López-Ferrer

Publications and source records attributed to Daniel López-Ferrer.

4 recordsLinked to original sources

PubMed2006

Mass spectrometry technologies for proteomics.

In the late 1980s, the advent of soft ionization techniques capable of generating stable gas phase ions from thermally unstable biomolecules, namely matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), laid the way for the development of a set of powerful alternatives to the traditional Edman chemistry for the structural characterization of peptides and proteins. The rapid protein identification capabilities that, coupled with two-dimensional gel electrophoresis, provided insights into all sorts of biological systems since the dawn of proteomics and have been exploited in the last few years for the development of more powerful and automatable gel-free strategies, mainly based on multidimensional chromatographic separations of peptides from proteolytic digests. In parallel to the evolution of ion sources, mass analysers and scan modes, the invention of new elegant biochemical strategies to fractionate or simplify highly complex mixtures, or to introduce isotopic labels in peptides in a variety of ways now makes also possible large-scale, high-coverage quantitative studies in a wide dynamic range. In this review, we provide the fundamental concepts of mass spectrometry (MS) and describe the technological progress of MS-based proteomics since its earliest days. Representative literature examples of their true power, either when employed as exploratory or as targeted techniques, is provided as well.

Mass Spectrometry↗

PubMed2006

Quantitative proteomics using 16O/18O labeling and linear ion trap mass spectrometry.

Quantitative proteomics using stable isotopic 16O/18O labeling has emerged as a very powerful tool, since it has a number of advantages over other methods, including the simplicity of chemistry, the constant mass tag at the C termini and its general applicability. However, due to the small mass difference between labeled and unlabeled peptide species, this approach has usually been restricted to high-resolution mass spectrometers. In this study we explored whether the high-resolution scanning mode, together with the extremely high scanning speed of the linear IT allows the 16O/18O-labeling method to be used for accurate, large-scale quantitative analysis of proteomes. A protocol, including digestion, desalting, labeling, MS and quantitative analysis was developed and tested using protein standards and whole proteome extracts. Using this method we were able to identify and quantify 140 proteins from only 10 mug of a proteome extract from mesenchymal stem cells. Relative expression changes larger than twofold can be identified with this method at the 95% confidence level. Our results demonstrate that accurate quantitative analysis using 16O/18O labeling can be performed in the practice using linear IT MS, without compromising large-scale peptide identification efficiency.

Cells, Cultured↗

PubMed2005

S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities.

Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-l-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.

Adenosine Triphosphatases↗

PubMed2004

Statistical model for large-scale peptide identification in databases from tandem mass spectra using SEQUEST.

Recent technological advances have made multidimensional peptide separation techniques coupled with tandem mass spectrometry the method of choice for high-throughput identification of proteins. Due to these advances, the development of software tools for large-scale, fully automated, unambiguous peptide identification is highly necessary. In this work, we have used as a model the nuclear proteome from Jurkat cells and present a processing algorithm that allows accurate predictions of random matching distributions, based on the two SEQUEST scores Xcorr and DeltaCn. Our method permits a very simple and precise calculation of the probabilities associated with individual peptide assignments, as well as of the false discovery rate among the peptides identified in any experiment. A further mathematical analysis demonstrates that the score distributions are highly dependent on database size and precursor mass window and suggests that the probability associated with SEQUEST scores depends on the number of candidate peptide sequences available for the search. Our results highlight the importance of adjusting the filtering criteria to discriminate between correct and incorrect peptide sequences according to the circumstances of each particular experiment.

Chromatography, Liquid↗