A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.endomembrane ͉ localization of organelle proteins by isotope tagging ͉ isotope tags for relative and absolute quantitation ͉ organelle proteomics P roteins are spatially organized according to their functions within the eukaryotic cell. Therefore, protein localization is an important step toward assigning functions to the thousands of uncharacterized proteins predicted by the genome-sequencing projects. Proteomics provides powerful tools for characterizing the protein contents of organelles. Confident protein localization, however, requires that either organelle preparations are free of contaminants or that techniques are used to discriminate between genuine organelle residents and contaminating proteins (1). Although reasonably pure preparations of some organelles, such as mitochondria, can be achieved, the components of the endomembrane system so far have proved recalcitrant to purification (2, 3). The constituent organelles of the endomembrane system have similar sizes and densities, making them difficult to separate. In addition, the proteins that reside within this system are in a constant state of flux. Endomembrane proteins traffic through the system en route to their final destination; for example, plasma membrane (PM) proteins travel although the endoplasmic reticulum (ER) and the Golgi apparatus before reaching the cell surface. Proteins within the endomembrane system also cycle between compartments; for example, ER residents continuously escape to the Golgi apparatus and are retrieved in COPI vesicles (4). Consequently, it is not sufficient merely to identify the proteins within a single organelle-enriched fraction. Instead, the steady-state distributions of proteins within the whole endomembrane system must be determined if a realistic insight into the subcellular localization of endomembrane proteins is to be achieved.Localization of organelle proteins by...

We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through selfgenerating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.

It is increasingly necessary to have a measuring instrument available in the health field that can be used in clinical practice and research. In order to guarantee the quality of their measurements it is essential that the instruments should be subjected to a process of validation. This process consists in adapting the instrument culturally to the setting where its psychometric characteristics are to be administered and checked, such as: reliability, validity, sensitivity and feasibility. There are measuring instruments from the health field available in other languages but that have not been validated into Spanish. Besides, the methodology for validating an instrument is little understood by the health professionals, which explains the indiscriminate use of instruments that have only been adapted or validated in a way that is not very consistent. The aim of this review is to bring up to date the process of validating an instrument for measuring health, and what it involves, in a practical way. The accessibility of evaluation instruments that have been culturally adapted and validated in different languages will facilitate the comparison of results obtained with the same instrument and the development international studies in different cultures.

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