RESUMEN
Phosphopeptide identification is still a challenging task because fragmentation spectra obtained by mass spectrometry do not necessarily contain sufficient fragment ions to establish with certainty the underlying amino acid sequence and the precise phosphosite. To improve upon this, it has been suggested to acquire pairs of spectra from every phosphorylated precursor ion using different fragmentation modes, for example CID, ETD, and/or HCD. The development of automated tools for the interpretation of these paired spectra has however, until now, lagged behind. Using phosphopeptide samples analyzed by an LTQ-Orbitrap instrument, we here assess an approach in which, on each selected precursor, a pair of CID spectra, with or without multistage activation (MSA or MS2, respectively), are acquired in the linear ion trap. We applied this approach on phosphopeptide samples of variable proteomic complexity obtained from Arabidopsis thaliana . We present a straightforward computational approach to reconcile sequence and phosphosite identifications provided by the database search engine Mascot on the spectrum pairs, using two simple filtering rules, at the amino acid sequence and phosphosite localization levels. If multiple sequences and/or phosphosites are likely, they are reported in the consensus sequence. Using our program FragMixer, we could assess that on samples of moderate complexity, it was worth combining the two fragmentation schemes on every precursor ion to help efficiently identify amino acid sequences and precisely localize phosphosites. FragMixer can be flexibly configured, independently of the Mascot search parameters, and can be applied to various spectrum pairs, such as MSA/ETD and ETD/HCD, to automatically compare and combine the information provided by these more differing fragmentation modes. The software is openly accessible and can be downloaded from our Web site at http://proteomics.fr/FragMixer.
Asunto(s)
Arabidopsis/metabolismo , Biología Computacional/métodos , Procesamiento Automatizado de Datos/métodos , Fosfopéptidos/aislamiento & purificación , Programas Informáticos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Arabidopsis/metabolismo , Secuencia de Consenso , Bases de Datos de Proteínas , Procesamiento Automatizado de Datos/instrumentación , Internet , Fosfopéptidos/metabolismo , Fosforilación , Proteómica/métodos , Motor de Búsqueda , Sensibilidad y Especificidad , Análisis de Secuencia de ProteínaRESUMEN
Within the growing field of proteomics, mass spectrometry is now established as a powerful tool for peptide and protein identification and discovery from purified samples. A new era is now beginning, with the development of MALDI imaging, maintaining the sensitivity and efficacy of both discovery and identification while additionally preserving the anatomical integrity of biomolecules like peptides, proteins, oligonucleotides and lipids within tissues. Crucial developments for sample preparations have made leaps and bounds, as it is now possible to work with freezed conserved biopsies (- 80 degrees c) of more than 6 months or even conserved after paraformaldehyde fixation and paraffin embedding. The latter development has opened the door to archived tissues in hospital libraries and biomarkers hunting from tissues derived from these libraries are now a key objective. The relationship between MALDI imaging and immunocytochemistry used by the pathologist is important. The development of specific MALDI imaging using probes with a tag (peptide or organic) called << Tag-Mass >> adds a whole new perspective. It is possible henceforth to localize a protein with its specific mRNA and more specifically, with its signalling pathway on the same sections or within a pathology expression phenotype from a biopsy. Development of such a technology is similar to the one that occurred several years ago for nuclear magnetic resonance (NMR) that leads the development of imaging technologies called MRI in hospital which is intensively used for pathology diagnostics.
Asunto(s)
Biomarcadores/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Animales , Criopreservación , Humanos , Técnicas para Inmunoenzimas , Proteínas/análisis , ARN Mensajero/análisis , Ratas , Reproducibilidad de los Resultados , Manejo de Especímenes , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta , Conservación de TejidoRESUMEN
MITICS is a new software developed for MALDI imaging. We tried to render this software compatible with all types of instruments. MITICS is divided in two parts: MITICS control for data acquisition and MITICS Image for data processing and images reconstruction. MITICS control is available for Applied BioSystems MALDI-TOF instruments and MITICS Image for both Applied BioSystems and Bruker Daltonics ones. MITICS Control provides an interface to the user for setting the acquisition parameters for the imaging sequence, namely set instruments acquisition parameters, create the raster of acquisition and control post-acquisition data processing, and provide this settings to the automatic acquisition software of the MALDI instrument. MITICS Image ensures image reconstruction, files are first converted to XML files before being loaded in a database. In MITICS image we have chosen to implement different data representations and calculations for image reconstruction. MITICS Image uses three different representations that have shown to ease extraction of information from the whole data set. It also offers image reconstruction base either on the maximum peak intensity or the peak area. Image reconstruction is possible for single ions but also by summing signals of different ions. MITICS was validated on biological cases.