Localization of Organelle Proteins by Isotope Tagging (LOPIT)

Abstract

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.

Quantitative Organelle Proteomics of MCF-7 Breast Cancer Cells Reveals Multiple Subcellular Locations for Proteins in Cellular Functional Processes

“…Our use of subfractionation of cellular organelles by sucrose gradient centrifugation is based on previous proteomics work indicating that fractionation of cytosol, plasma membrane, endoplasmic reticulum, Golgi and mitochondria is readily obtained 22 and that with more sophisticated analysis of protein distribution along the gradient, as many as 10 subcellular locations can be distinguished. 23 The subsequent steps in identifying the proteins present in different fractions of the sucrose gradient (1D SDS gels, gel slicing, proteolytic production of peptides, and identification of peptides by MS) are standard proteomics techniques, and our use of them is described in detail in the Materials and Methods section.…”

“…The main disadvantages of this approach are that the degree of purification/ contamination of the organelle is difficult to ascertain conclusively for lower abundance proteins, that the protein content may be altered by the purification process and that the approach is not very suitable for dynamic studies of protein subcellular location. In a few cases, 22,23 an alternative approach of partial purification of organelles in a sucrose gradient has been employed, but the assignment of proteins to individual organelles has been based on matching gradient profiles of proteins to the profiles of presumptive marker proteins. This is useful for identifying what might be denominated core proteins of an organelle, but is automatically biased against evaluation of proteins in multiple subcellular locations.…”

Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

“…It contains a large and diverse set of glycosyltransferases and other enzymes that are required for the synthesis and modification of these polysaccharides (Parsons et al, 2012b;Oikawa et al, 2013). The protein composition of this organelle has been the focus of a number of studies; however, these studies largely report a catalog of Golgi-localized proteins, and to date, there are no comprehensive data on the relative abundance of the different protein constituents of the Golgi apparatus (Dunkley et al, 2004Sadowski et al, 2008;Nikolovski et al, 2012;Groen et al, 2014). The quantification of the plant Golgi proteome has been considered challenging, because this organelle is proportionally of low abundance in the cell; therefore, its constituent proteins are rarely identified in conventional proteomics experiments.…”

A “Tagless” Strategy for Identification of Stable Protein Complexes Genome-wide by Multidimensional Orthogonal Chromatographic Separation and iTRAQ Reagent Tracking

“…Concurrently, the parent polypeptide's relative abundance, within the analyzed fraction set, is calculated based on the intensities of four iTRAQ reporter ions. A similar approach of using either iCAT or iTRAQ reagent-labeling to follow protein gradient distribution profiles under conditions of sedimentation was recently introduced by Kathryn Lilley et al [30][31][32] A Proof-of-Principle Demonstration. To provide a first proof-of-principle that iTRAQ quantification allows protein complexes to be detected, we first performed a minimal twostep chromatographic separation of an E. coli crude extract, which takes high molecular weight proteins derived from a size exclusion column and fractionates them further by Mono Q anion exchange chromatography.…”