Each population of organelles is characterized by size, density, charge, and other properties on which the separation relies [192] . Analyzing subcellular fractions and organelles allows tracking proteins that shuttle between different compartments, for example, between the cytoplasm and nucleus. A high dynamic range of proteins can be partially achieved by fractionation of the proteome into subproteomes by applying affinity purification may allow proteomic analysis of low copy number proteins [193] . The nuclear, chloroplast, amyloplast, plasma membrane, peroxisome, endoplasmic reticulum, cell wall, and mitochondrial proteomes were successfully characterized in Arabidopsis [194] . Several groups have taken advantage of this approach to recover a higher percentage of membrane proteins from subcellular extracts using various nonionic and zwitterionic detergents or phase-partitioning methods. These efforts resulted in the successful determination of the protein complement of the thylakoid and envelope membrane systems of the chloroplast [195] . By enriching for the protein class of interest based on a particular chemical/physical characteristic(s), offer the advantage of reducing sample complexity and access to lower abundance proteins in a discoverydriven experimental approach [196] . Free flow electrophoresis (FFE) utilizes differences in electrophoretic mobility rather than density to separate cells or subcellular organelles [197] . FFE has previously been used in separating endosomes from hamster ovary cells [198] , plasma membrane from human platelets [199] , and insulin transporting vesicles in liver cells. The separation is based on the electrophoretic motility of cells or cell organelles suspended in a vertical free flowing buffer film on which an electric field is applied at a right angle to the flow direction. FFE has been a most valuable tool in the investigation of the composition of secretory vesicles and in addition, it has clarified how the membrane of plasma membrane vesicles is oriented after nitrogen disruption of human neutrophils [200] . Importantly, subcellular fractionation is a flexible and adjustable approach that may be efficiently combined not only with 2D gel electrophoresis but also with gelindependent techniques. However, they do have limitations of considerable cross-contamination with other subcellular organelles.