Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy

Visualisation of multiple isoforms of kappa-casein on 2-D gels is restricted by the abundant alpha- and beta-caseins that not only limit gel loading but also migrate to similar regions as the more acidic kappa-casein isoforms. To overcome this problem, we took advantage of the absence of cysteine residues in alpha(S1)- and beta-casein by devising an affinity enrichment procedure based on reversible biotinylation of cysteine residues. Affinity capture of cysteine-containing proteins on avidin allowed the removal of the vast majority of alpha(S1)- and beta-casein, and on subsequent 2-D gel analysis 16 gel spots were identified as kappa-casein by PMF. Further analysis of the C-terminal tryptic peptide along with structural predictions based on mobility on the 2-D gel allowed us to assign identities to each spot in terms of genetic variant (A or B), phosphorylation status (1, 2 or 3) and glycosylation status (from 0 to 6). Eight isoforms of the A and B variants with the same PTMs were observed. When the casein fraction of milk from a single cow, homozygous for the B variant of kappa-casein, was used as the starting material, 17 isoforms from 13 gel spots were characterised. Analysis of isoforms of low abundance proved challenging due to the low amount of material that could be extracted from the gels as well as the lability of the PTMs during MS analysis. However, we were able to identify a previously unrecognised site, T(166), that could be phosphorylated or glycosylated. Despite many decades of analysis of milk proteins, the reasons for this high level of heterogeneity are still not clear.     

Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry

Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.     

Proteomic analysis of kappa-casein micro-heterogeneity

The proteome of bovine milk is dominated by just six gene products that constitute approximately 95% of milk protein. Nonetheless, over 150 protein spots can be readily detected following two-dimensional electrophoresis of whole milk. Many of these represent isoforms of the major gene products produced through extensive post-translational modification. Peptide mass fingerprinting of in-gel tryptic digests (using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) in reflectron mode with alpha-cyano-4-hydroxycinnamic acid as the matrix) identified 10 forms of kappa-casein with isoelectric point (pI) values from 4.47 to 5.81, but could not distinguish between them. MALDI-TOF MS in linear mode, using sinapinic acid as the matrix, revealed a large tryptic peptide (mass > 5990 Da) derived from the C-terminus that contained all the known sites of genetic variance, phosphorylation and glycosylation. Two genetic variants present as singly or doubly phosphorylated forms could be distinguished using mass data alone. Glycoforms containing a single acidic tetrasaccharide were also identified. The differences in electrophoretic mobility of these isoforms were consistent with the addition of the acidic groups. While more extensively glycosylated forms were also observed, substantial loss of N-acetylneuraminic acid from the glycosyl group was evident in the MALDI spectra such that ions corresponding to the intact glycopeptide were not observed and assignment of the glycoforms was not possible. However, by analysing the pI shifts observed on the two-dimensional gels in conjunction with the MS data, the number of N-acetylneuraminic acid residues, and hence the glycoforms present, could be determined.     

Proteomic changes in the heart of diet-induced pre-diabetic mice

The development of type 2 diabetes (T2D) is strongly associated with obesity. In humans, T2D increases the risk for end organ complications. Among these, heart disease has been ranked as the leading cause of death. We used a proteomic methodology to test the hypothesis that a pre-diabetic state generated by high-fat diet leads to changes in proteins related to heart function and structure. Over 300 protein spots were resolved by two-dimensional gel electrophoresis (2-DE). Fifteen protein spots were found to be altered (7 decreased and 8 increased) in pre-diabetic hearts. The protein spots were then identified by mass spectrometry and immunoblots. Among the decreased proteins, 3 are involved in heart structure (one isoform of desmin, troponin T2 and α-cardiac actin), 3 are involved in energy metabolism (mitochondrial ATP synthase β subunit, adenylate kinase and creatine kinase) and one is a component of the citric acid cycle (isocitrate dehydrogenase 3). In contrast, proteins involved in fatty acid oxidation (two isoforms of peroxisomal enoyl-CoA hydratase) and the citric acid cycle (three isoforms of malate dehydrogenase) were increased in pre-diabetic hearts. The results suggest that changes in the levels of several heart proteins may have implications in the development of the cardiac phenotype associated to T2D.     

Analysis of the human casein phosphoproteome by 2-D electrophoresis and MALDI-TOF/TOF MS reveals new phosphoforms

Mammalian breast milk contains an array of proteins and other nutrients essential for the development of the newborn. In human milk, the caseins (alpha S1, beta and kappa) are a major class of proteins; however, the dynamic range of concentrations in which the various isoforms of each casein exist presents challenges in their characterization. To study human milk casein phosphoforms, we applied traditional two-dimensional polyacrylamide gel electrophoretic (2-DE) separation combined with matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) tandem mass spectroscopic analysis. The abundant beta-casein was resolved as a train of 6 spots differing in phosphorylation level with 0-5 phosphates attached. To study the less abundant alpha S1-casein, a cysteine-tagging enrichment treatment was used prior to 2-DE. A train of 9 spots with 4.4 < p I < 5.3 were identified as alpha S1-casein. This included five previously uncharacterized phosphoforms with up to 8 phosphate groups located in two serine-rich tryptic phosphopeptides ( (27)L-R (51), (69)N-K (98)) consistent with alpha-caseins from various ruminant species. MS/MS analysis of the phosphopeptides released by tryptic digestion enabled identification of the residue-specific order of phosphorylation among the 6 beta-casein and 9 alpha S1-casein phosphoforms. Deamidation of N (47) of alpha S1-casein was also a feature of the MS analysis. This study represents the first comprehensive analysis of the human casein phosphoproteome and reveals a much higher level of phosphorylation than previously recognized. It also highlights the advantages of 2-DE for examining the global pattern of protein phosphoforms and the limitations of attempting to estimate phosphorylation site occupancies from "bottom-up" studies.     

Characterisation of host defence proteins in milk using a proteomic approach

Besides providing nutrition to the newborn, milk also protects the neonate and the mammary gland against infection. As well as the six major proteins, bovine milk contains minor proteins, not all of which have been characterized. In this study, we have subjected bovine skim milk, whey, and milk fat globule membrane (MFGM) fractions to both direct liquid chromatography-tandem mass spectrometry (LC-MS/MS), and two-dimensional electrophoresis (2-DE) followed by matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) of individual protein spots to better characterize the repertoire of minor milk proteins, particularly those involved with host defense. Milk from peak lactation as well as during the period of colostrum formation and during mastitis were analyzed to gain a more complete sampling of the milk proteome. In total, 2903 peptides were detected by LC-MS and 2770 protein spots by 2-DE. From these, 95 distinct gene products were identified, comprising 53 identified through direct LC-MS/MS and 57 through 2-DE-MS. The latter were derived from a total of 363 spots analyzed with 181 being successfully identified. At least 15 proteins were identified that are involved in host defense. These results demonstrate that the proteome of milk is more complex than has previously been reported and a significant fraction of minor milk proteins are involved in protection against infection.     

Spatio-temporal profiling and degradation of alpha-amylase isozymes during barley seed germination

Ten genes from two multigene families encode barley alpha-amylases. To gain insight into the occurrence and fate of individual isoforms during seed germination, the alpha-amylase repertoire was mapped by using a proteomics approach consisting of 2D gel electrophoresis, western blotting, and mass spectrometry. Mass spectrometric analysis confirmed that the 29 alpha-amylase positive 2D gel spots contained products of one (GenBank accession gi|113765) and two (gi|4699831 and gi|166985) genes encoding alpha-amylase 1 and 2, respectively, but lacked products from seven other genes. Eleven spots were identified only by immunostaining. Mass spectrometry identified 12 full-length forms and 12 fragments from the cultivar Barke. Products of both alpha-amylase 2 entries co-migrated in five full-length and one fragment spot. The alpha-amylase abundance and the number of fragments increased during germination. Assessing the fragment minimum chain length by peptide mass fingerprinting suggested that alpha-amylase 2 (gi|4699831) initially was cleaved just prior to domain B that protrudes from the (betaalpha)(8)-barrel between beta-strand 3 and alpha-helix 3, followed by cleavage on the C-terminal side of domain B and near the C-terminus. Only two shorter fragments were identified of the other alpha-amylase 2 (gi|166985). The 2D gels of dissected tissues showed alpha-amylase degradation to be confined to endosperm. In contrast, the aleurone layer contained essentially only full-length alpha-amylase forms. While only products of the above three genes appeared by germination also of 15 other barley cultivars, the cultivars had distinct repertoires of charge and molecular mass variant forms. These patterns appeared not to be correlated with malt quality.     

Study of posttranslational modifications in lenticular alphaA-Crystallin of mice using proteomic analysis techniques

In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.     

Evidence for isoforms of the phosphorylatable myosin light chain in rat uterus

The phosphorylatable myosin light chain of rat uterus was resolved into four spots by two-dimensional gel electrophoresis. Antibody against the 20 kDa light chain from smooth muscle myosin recognized these four spots. Purified light chain exhibited four spots on a diagonal upon isoelectrofocusing in both first and second dimensions, proving that these spots are not due to artifactual charge modification. Accordingly, the four spots contain light chain isoforms derived from myosin.     

Characterization and localization of tropomyosin proteins in Xenopus embryos with specific antibodies

In the process of monoclonal antibody (mAb) production against the 38kDa protein which is lacking in the gastrula arrested mutant embryos in Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) proteins in Xenopus embryos. The characterization of the corresponding antigens to those mAb was performed by immunoblotting and silver staining for two-dimensional (2-D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescent microscopy.
By 2-D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid-tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole.
Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms.     

Development of narrow immobilized pH gradients covering one pH unit for human seminal plasma proteomic analysis

Micropreparatively loaded two-dimensional (2-D) electrophoresis gels were used to identify human seminal plasma polypeptides by using narrow immobilized pH gradients (IPGs) covering one pH unit as first dimension. This investigation was restricted to IPG 4.5-5.5 and 5-6 because of the main spot distribution in the acidic part of the 2-D map performed with IPG 3-10, a zone presumed to be rich in spermatogenic markers. Both highly expressed and minor spots of the 2-D map were analyzed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectroscopy analysis. Identification was obtained by a combination of mass spectrometry and database searching. Identified proteins were of different origin from the male genital tract and some proteolysis was observed. They appeared as either isolated molecules or isoforms. At the analytical level, narrow IPGs allowed a two-fold increase in the number of spots, improved resolution of minor spots particularly in surrounding of highly expressed spots and sensitivity level. Some of these faint spots were differently expressed in azoospermic patients as compared to normospermic men. Therefore, zooming-in on the proteome of human seminal plasma allowed more accurate differential expression analysis of impaired spermatogenesis associated markers.     

Proteomic analysis of the 14-3-3 family in Arabidopsis

In this study, various proteomics-based methods were utilized to examine the 14-3-3 protein family in Arabidopsis thaliana. A protein extract was prepared from an Arabidopsis hypocotyl suspension culture and analyzed by two-dimensional gel electrophoresis and immunoblotting with a 14-3-3 monoclonal antibody that recognizes multiple Arabidopsis isoforms. Protein spots that cross-reacted with the monoclonal antibody as well as the surrounding spots were analyzed by high performance liquid chromatography in conjunction with electrospray-tandem mass spectrometry. Nine separate spots contained 14-3-3s and each spot contained multiple 14-3-3 isoforms. Every isoform observed was verified by the identification of at least one isoform-specific peptide. Further analysis by mass spectrometry revealed that the isoforms Chi, Upsilon, Omega, Phi, and Lambda were acetylated on their N termini and no non-acetylated N termini were recovered. These data, together with the distribution of isoforms and the confirmation that 14-3-3s are not complexed during urea denaturing isoelectric focusing, supports the conclusion that Arabidopsis 14-3-3s are acetylated in vivo and are significantly affected by other post-translational modifications.     

Strategic proteome analysis of Candida magnoliae with an unsequenced genome

Erythritol is a noncariogenic, low calorie sweetener. It is safe for people with diabetes and obese people. Candida magnoliae is an industrially important organism because of its ability to produce erythritol as a major product. The genome of C. magnoliae has not been sequenced yet, limiting the available proteome database. Therefore, systematic approaches were employed to construct the proteome map of C. magnoliae. Proteomic analysis with systematic approaches is based on two-dimensional electrophoresis, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), tandem mass spectrometry (MS/MS) and database interrogation. First, 24 spots were analyzed using peptide mass fingerprinting along with MALDI-TOF MS with high mass accuracy. Only four spots were reliably identified as carbonyl reductase and its isoforms. The reason for low sequence coverage seemed to be that these identification strategies were based on the presence of the protein database obtained from the publicly accessible genome database and the availability of cross-species protein identification. MS/MS (MS/MS ion search and de novo sequencing) in combination with similarity searches allowed successful identification of 39 spots. Several proteins including transaldolase identified by MS/MS ion searches were further confirmed by partial sequences from the expressed sequence tag database. In this study, 51 protein spots were analyzed and then potentially identified. The identified proteins were involved in glycolysis, stress response, other essential metabolisms and cell structures.     

Changes in protein expression profiles in bivalve molluscs (Chamaelea gallina) exposed to four model environmental pollutants

Proteomics has been used in the clam Chamaelea gallina as a preliminary screening of changes in protein expression caused by pollutants, potentially useful as new biomarkers. Clams were exposed in water for seven days to four model contaminants, Aroclor 1254, copper(II), tributyltin (TBT), and arsenic(III), and cytosolic fractions were initially analyzed by two-dimensional (2-D) electrophoresis in 7 cm IPG strips (pH 4-7). On average, about 1000 spots were resolved and altered expression was qualitatively detected in 9-26 spots per treatment. Aroclor 1254, Cu(II) and As(III) had a mainly upregulating effect, in contrast to TBT. Altered protein expression was confirmed in 18 cm gels (at narrow pH ranges). The 15 spots more drastically altered were excised and analyzed by mass spectrometry (MS), and four proteins were identified. Aroclor 1254 and Cu(II) upregulated putative isoforms of tropomyosin and light chain of myosin. Actin was downregulated by Aroclor and Cu(II) but upregulated by TBT and As(III), while the opposite behavior was shown by a truncated actin form, homologous to the Drosophila act87E gene product. The exclusive identification of cytoskeletal proteins could reflect their relative abundance, their prevalence in databases in molluscs, or their role as major targets of pollutant-related oxidative stress.     

Comparative proteomic analysis for hCTLA4Ig production in transgenic rice suspension cultures using two-dimensional difference gel electrophoresis

The new technology, two-dimensional difference gel electrophoresis (2D DIGE), uses fluorescent dyes to simplify the process of detecting and matching proteins between multiple gels by allowing for the separation of up to three separate protein samples within the same gel. In this study, recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4lg) was produced in transgenic rice suspension cell cultures and the intracellular proteins were analyzed by 2D DIGE. The highest level of hCTLA4Ig (25.4 mg/L) was obtained five days after induction. The intracellular proteins expressed at both the growth and induction culture stages were separated and analyzed using DeCyder software. At least 2,218 spots were detected with two-fold thresholds and 95% confidence. We found that 29 spots increased and 20 spots decreased in their intensities during the production of recombinant hCTLA4Ig. In addition, the 2D Western blot of hCTLA4Ig revealed that this fusion protein was expressed in a variety of isoforms.     

Proteomic and genomic characterization of Kunitz trypsin inhibitors in wild and cultivated soybean genotypes

In this study, we investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), the cultivated soybean (G. max) ancestors of modern N. American soybean cultivars (old), modern N. American soybean (elite), and Asian cultivated soybean landraces that were the immediate results of domestication from the wild soybean. Proteins were well separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and stained protein cut from a 2D-PAGE indicated that KTI exists as multiple isoforms (spots) in soybean. Protein spots of KTI were identified and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although overall distribution patterns of the KTI protein spots appeared similar, the number and intensity of the protein spots between wild and cultivated genotypes varied. Three KTI peptides were identified in three of the wild genotypes, PI 393551, PI 407027 and PI 407282, in which KTI3 peptide showed highest intensity. The remaining wild genotype, PI 366120, showed four protein spots. In contrast, the ancestors, modern and Asian landrace genotypes showed only two protein spots corresponding to KTI. On the basis of DNA blot analysis, there is one copy of the KTI3 gene in all 16 genotypes. Polymorphism was detected in one of the wild genotypes (PI 366120) both in proteomic and genomic analyses. Our data suggest that the major variation of protein profiles were between wild and cultivated soybean genotypes rather than among genotypes in the same group. Genetic variation of KTI1, KTI2 and KTI3-related genes were detected within and between groups.     

Three isoforms of cyclophilin A associated with human immunodeficiency virus type 1 were found by proteomics by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry

Human immunodeficiency virus type 1 (HIV-1) strain LAV-1 (HIV-1(LAV-1)) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified HIV-1(LAV-1) particles was subjected to two-dimensional (2D) gel electrophoresis and stained. The 2D gel electrophoresis image suggested that 24 proteins can be identified inside the virion. Furthermore, the stained protein spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Peptide mass fingerprinting data suggested that two isoforms of cyclophilin A (CyPA), one with an isoelectric point (pI) of 6.40 and one with a pI of 6.53, are inside the viral membrane; that another isoform, with a pI of 6.88, is outside the viral membrane; and that the CyPA isoform with a pI of 6.53 is N acetylated. The mechanisms that permit the redistribution of CyPA on the viral surface have not yet been clarified, but it is surmised that the CyPA isoform with a pI of 6.88 may play a critical role in the attachment of virions to the surface of target cells and that both CyPA isoforms with pIs of 6.40 and 6.53 may regulate the conformation of the HIV-1 capsid protein.     

Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins

Extraction of soybean seed proteins for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry analysis is challenging and inconsistent. In this study, we compared four different protein extraction/solubilization methods-urea, thiourea/urea, phenol, and a modified trichloroacetic acid (TCA)/acetone-to determine their efficacy in separating soybean seed proteins by 2D-PAGE. In all four methods, seed storage proteins were well separated by 2D-PAGE with minor variations in the intensity of the spots. The thiourea/urea and TCA methods showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, several less abundant and high molecular weight proteins were clearly resolved and strongly detected using the thiourea/urea and TCA methods. Protein spots obtained from the TCA method were subjected to mass spectrometry analysis to test their quality and compatibility. Fifteen protein spots were selected, digested with trypsin, and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS). The proteins identified were beta-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gly m Bd 28K allergen, and sucrose binding proteins. These results suggest that the thiourea/urea and TCA methods are efficient and reliable methods for 2D separation of soybean seed proteins and subsequent identification by mass spectrometry.     

Prediction of translocation and cleavage of heterogeneous ribonuclear proteins and Rho guanine nucleotide dissociation inhibitor 2 during apoptosis by subcellular proteome analysis

Jurkat T cells induced to undergo apoptosis by the CD95(Fas/Apo-1) pathway were investigated by proteome analysis. The most prominent differing protein spots of apoptotic and nonapoptotic cells were identified as various heterogeneous ribonuclear proteins (hnRNPs) and Rho guanin nucleotide dissociation inhibitor (GDI) 2. In apoptotic cells, four spots slightly differing in molecular mass and/or isoelectric point were identified as Rho GDI 2 with the mass and pI as expected after caspase-3 cleavage near the N-terminus. Subcellular proteome analysis revealed that Rho GDI 2 was highly enriched in the cytosolic fraction, present in minor amounts in the nuclear fraction and absent from the mitochondrial fraction. In apoptotic cells however, the spots representing processed and modified Rho GDI 2 were found in the cytosol, in the nucleus and also the mitochondria at different spot positions. In addition, twelve different hnRNPs were identified to be altered after induction of cell death of which hnRNPs A/B, D, F, H, I and L were hitherto unknown to be modified during apoptosis. Most of the hnRNP spots were found in the nucleus of nonapoptotic cells, whereas these proteins, either modified or unmodified, relocated to the cytosol and/or the mitochondria in apoptotic cells. Our results demonstrate that modification of proteins during apoptosis is often accompanied by their relocalisation between cellular compartments.