Pre-analytical operating procedures for serum Low Molecular Weight protein profiling.
ABSTRACT Biological specimen collection and storage are an integral component of serum proteomics research. Although many efforts have been posed to address the effects of pre-analytical procedures, standardized protocols for collection and storage of samples for Low Molecular Weight (LMW) proteome profiling are still needed. Here we report a systematic analysis on the influence of pre-analytical factors [clotting times, temperature and time storage, addition of protease inhibitor (PI)] on serum LMW proteome profiling. Moreover, a comparison between manual versus automated peptide purification by functionalized magnetic bead-based MALDI-MS approach was performed. The results demonstrated best serum LMW proteins recovery and stability using a clotting time between 1 and 2h, with serum stored up to 2h either at room temperature or at 4 degrees C, independently of PI addition. PI addition to whole blood resulted in a lower number of LMW peaks detected. Finally, minimal effects on serum proteome profiles were observed after 1-month storage at -80 degrees C, independently of PI addition on whole blood and/or serum. In conclusion, the use of standardized pre-analytical and storage procedures together with an automated peptide purification might minimize potential bias on serum LMW profiling results, thus allowing a better homogeneity and reproducibility in future proteomics studies.
- [Show abstract] [Hide abstract]
ABSTRACT: Mass spectrometry platforms have attracted a lot of interest in the last 2 decades as profiling tools for native peptides and proteins with clinical potential. However, limitations associated with reproducibility and analytical robustness, especially pronounced with the initial SELDI systems, hindered the application of such platforms in biomarker qualification and clinical implementation. The scope of this article is to give a short overview on data available on performance and on analytical robustness of the different platforms for peptide profiling. Using the CE-MS platform as a paradigm, data on analytical performance are described including reproducibility (short-term and intermediate repeatability), stability, interference, quantification capabilities (limits of detection), and inter-laboratory variability. We discuss these issues by using as an example our experience with the development of a 273-peptide marker for chronic kidney disease. Finally, we discuss pros and cons and means for improvement and emphasize the need to test in terms of comparative clinical performance and impact, different platforms that pass reasonably well analytical validation tests.Clinical biochemistry 10/2012; · 2.02 Impact Factor
Article: Repressors of photomorphogenesis.[Show abstract] [Hide abstract]
ABSTRACT: The internal programs of plant development are informed in a profound way by environmental light conditions. This review summarizes the contribution of repressor proteins to the light-signaling machinery during seedling development, and discusses the integration of repressors with other, positively acting, light-signaling pathways and auxin and brassinosteroid hormone-signaling pathways. The main focus is placed on the mode of action of the COP/DET/FUS proteins, which were first identified in Arabidopsis but are now emerging in other plants. Their role in regulating protein turnover through ubiquitination is reviewed in light of parallel ongoing investigations of COP/DET/FUS homologues in metazoans and fungi.International Review of Cytology 02/2002; 220:185-223. · 6.09 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The development of Biobanks and recent advances in molecular biology have enhanced the possibility to accelerate translational research studies. The Interinstitutional Multidisciplinary BioBank (BioBIM) is organized in a large healthy donors collection and pathology-based biobanks with the aim to provide a service for development of interdisciplinary studies. A new pathology-based biobank has been organized to specifically collect biospecimen from patients affected by migraine, with the final goal to centralize data, collect blood, plasma, serum, DNA and RNA of patients with this disease. The BioBIM is fully equipped for the automation of sampling/processing, storage and tracking of biospecimens. Standard Operating Procedures have been developed for processing and storage phases as well as archive of clinical data. The availability of biospecimens and clinical data will constitute a resource for various research projects.Neurological Sciences 01/2013; · 1.41 Impact Factor