Table 1.

Examples of organelle isolation and subproteome analysis in plants.

Materials	Organelles	Isolation protocols	Purity tests	Marker proteins	Protein extraction	Proteomic methods	Notes	References*
Posidonia oceanic leaves	Chloroplast	Differential centrifugation, Percoll and sucrose gradient centrifugation	Enzyme activities, immunoblotting	Coproporphyrinogen III oxidase	Phenol extraction	SDS-PAGE, HPLC-Chip/MS	43 chloroplast proteins identified	Piro et al., 2015
Arabidopsis leaves	Chloroplast	Differential centrifugation, Percoll gradient centrifugation	Fluorescence microscopy, immunoblotting, enzyme activities	Fumarase, catalase	Stepwise extraction using Tris-HCl buffer, 2DE rehydration medium and 4% SDS solution	SDS-PAGE, LC/ MS/MS	690 proteins identified from purified chloroplasts	Kleffmann et al., 2004
Chickpea seeds	Chloroplast	Filtration, Percoll gradient centrifugation	Chlorophyll determination, immunoblotting, enzyme activities	LHCII, CPN 60, TOC 75, CoxII, V-ATPase	7M urea solution extraction	SDS PAGE, LC-MS/MS	2451 proteins identified	Lande et al., 2017
Brassica napus embryos	Plastid	Filtration with different size sieve	As above	Biotin carboxylase carrier protein	0.5 × SDS-PAGE buffer extraction	SDS-PAGE, Semi-continuous MudPIT MS/MS	Often being referred	Jain et al., 2008
Medicago truncatula roots	Plastid	Differential centrifugation, filtration, gradient centrifugation	Nitrite reductase activity, immunoblotting	Cytosolic UDP-Glucose pyrophosphatase	Acetone precipitation	SDS-PAGE, GeLC-MS/MS	266 proteins identified	Daher et al., 2010
Wheat kernels	Starch granule	CsCl gradient centrifugation	Electron microscopy	α-glucan phosphorylase	TCA/acetone precipitation and phenol extraction	2D-GE, peptide mass fingerprinting and LC-MS/MS	85 proteins identified	Bancel et al., 2010
Rice endosperm	Starch granule	Filtration with different size sieves	I2 staining, microscopy, immunoblotting	α-glucan phosphorylase	Phenol extraction	SDS-PAGE, LC-MS/MS	1157 proteins identified	Xing et al., 2016
Wheat florets	Mitochondrion	Differential centrifugation, filtration, Percoll gradient centrifugation	Enzyme activities, electron microscopy, 0.01 M Janus green B staining	Cytochrome c oxidase	2DE rehydration buffer extraction	2D-GE, LC-MS/MS	71 proteins identified	Wang et al., 2015
Potato tubers	Mitochondrion	AS above	Immunoblotting	Enolase, plastidic α-carboxytransferase, mitochondrial PDE1-α	SDS extraction	SDS-PAGE, GeLC-MS/MS	1060 proteins identified	Salvato et al., 2014
Arabidopsis suspension cells	Vacuole	Differential centrifugation, Percoll and sucrose gradient centrifugation	Enzyme activities, immunoblotting	V-type H+-ATPase	SDS extraction	SDS-PAGE, LC-MS/MS	163 proteins identified; often being referred	Shimaoka et al., 2004
Tobacco, potato, apple leaves	Nucleus	Filtration, differential centrifugation, Percoll and sucrose gradient centrifugation	DAPI staining, microscopy, immunoblotting	OsRH36-GFP	TRizol reagent extraction	SDS-PAGE	none	Sikorskaite et al., 2013
Arabidopsis suspension cells	Golgi apparatus	c-Myc tag; iodixanol density ultra-centrifugation	Immunoblotting	Gtl6/At2g29900	TCA/acetone precipitation	LC-IMS-MS	102 Golgi-localized proteins identified	Nikolovski et al., 2014
AS above	Golgi apparatus	Sucrose gradient centrifugation	Immunoblotting, electron microscopy, enzyme activities	NDPase	FFE buffers extraction	SDS-PAGE, LC-MS/MS	371 proteins identified	Parsons et al., 2012
Castor seeds	Endoplasmic reticulum	Differential centrifugation, filtration, sucrose gradient centrifugation	MS/MS identification	Oleate-12-hydroxylase	DOC-TCA precipitation	SDS-PAGE, 2D-GE, MALDI-TOF-MS, Q-TOF MS/MS	300 proteins identified, often being referred	Maltman et al., 2002
Arabidopsis leaves	Peroxisome	AS above	Enzyme activities, MS/MS identification	Hydroxypyruvate reductase	2DE rehydration buffer extraction	2D-GE, LC-MS/MS	Often referred	Reumann et al., 2007
Arabidopsis hypocotyls	Cell wall	Filtration, differential centrifugation	LC-MS/MS identification	N-glycoprotein	Salt/SDS buffer extraction	2D-GE, LC-MS/MS	Often referred	Feiz et al., 2006
Alfalfa stems	Cell wall	Low-salt/gradient centrifugation	As above	N-glycoprotein	EGTA/LiCl solution extraction and methanol/chloroform precipitation	SDS-PAGE, LC-MS/MS	245 proteins identified	Verdonk et al., 2012

These studies were selected because the isolation protocols allowed for high-purity of specific organelles and the large number of organelle proteins identified by subproteomics analysis.