Regulation of the localisation and function of the oncogene LYRIC/AEG-1 by ubiquitination at K486 and K491.

The pivotal role of LYRIC/AEG-1 in malignant transformation, tumourigenesis and chemo-resistance has previously been demonstrated in different cell types and sub-cellular compartments. The localisation of LYRIC/AEG-1 appears crucial to its function and is regulated by three lysine-rich nuclear localisation signal regions, one of which was previously demonstrated to be modified by ubiquitin. Here we show that mutation of LYRIC/AEG-1 at K486 and K491 results in a loss of ubiquitination. A K486/491R double mutant that is incapable of ubiquitination shows reduced binding to the NFκB subunit p65 or importin-ß resulting in a distinctive peri-nuclear localisation of LYRIC/AEG-1. We also provide evidence to suggest that TOPORS, an E3 ligase that also regulates p53 modification may be responsible for LYRIC/AEG-1 ubiquitin modification. Overall we demonstrate that specific sites of LYRIC/AEG-1 ubiquitination are essential for regulating LYRIC/AEG-1 localisation and functionally interacting proteins.

Functional proteomic analysis of long-term growth factor stimulation and receptor tyrosine kinase coactivation in Swiss 3T3 fibroblasts.

In Swiss 3T3 fibroblasts, long-term stimulation with PDGF, but not insulin-like growth factor 1 (IGF-1) or EGF, results in the establishment of an elongated migratory phenotype, characterized by the formation of retractile dendritic protrusions and absence of actin stress fibers and focal adhesion complexes. To identify receptor tyrosine kinase-specific reorganization of the Swiss 3T3 proteome during phenotypic differentiation, we compared changes in the pattern of protein synthesis and phosphorylation during long-term exposure to PDGF, IGF-1, EGF, and their combinations using 2DE-based proteomics after (35)S- and (33)P-metabolic labeling. One hundred and five differentially regulated proteins were identified by mass spectrometry and some of these extensively validated. PDGF stimulation produced the highest overall rate of protein synthesis at any given time and induced the most sustained phospho-signaling. Simultaneous activation with two or three of the growth factors revealed both synergistic and antagonistic effects on protein synthesis and expression levels with PDGF showing dominance over both IGF-1 and EGF in generating distinct proteome compositions. Using signaling pathway inhibitors, PI3K was identified as an early site for signal diversification, with sustained activity of the PI3K/AKT pathway critical for regulating late protein synthesis and phosphorylation of target proteins and required for maintaining the PDGF-dependent motile phenotype. Several proteins were identified with novel PI3K/Akt-dependent synthesis and phosphorylations including eEF2, PRS7, RACK-1, acidic calponin, NAP1L1, Hsp73, and fascin. The data also reveal induction/suppression of key F-actin and actomyosin regulators and chaperonins that enable PDGFR to direct the assembly of a motile cytoskeleton, despite simultaneous antagonistic signaling activities. Together, the study demonstrates that long-term exposure to different growth factors results in receptor tyrosine kinase-specific regulation of relatively small subproteomes, and implies that the strength and longevity of receptor tyrosine kinase-specific signals are critical in defining the composition and functional activity of the resulting proteome.

Site recognition and substrate screens for PKN family proteins.

The PRKs [protein kinase C-related kinases; also referred to as PKNs (protein kinase Ns)] are a kinase family important in diverse functions including migration and cytokinesis. In the present study, we have re-evaluated and compared the specificity of PKN1 and PKN3 and assessed the predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and PKN3 phosphorylate serine residues in sequence contexts that have an arginine residue in position -3. In contrast, PKN1 and PKN3 do not tolerate arginine residues in position +1 and -1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP-170 (cytoplasmic linker protein of 170 kDa); a PKN target site was identified that conformed to the predicted pattern. Using a protein array, we identified 22 further substrates for PKN1, of which 20 were previously undescribed substrates. To evaluate further the recognition signature, the site on one of these hits, EGFR (epidermal growth factor receptor), was identified. This identified Thr654 in EGFR as the PKN1 phosphorylation site and this retains an arginine residue at the -3 position. Finally, the constitutive phosphorylation of EGFR on Thr654 is shown to be modulated by PKN in vivo.

Mammal-restricted elements predispose human RET to folding impairment by HSCR mutations.

The maturation of human RET is adversely affected by a range of missense mutations found in patients with Hirschsprung's disease (HSCR), a complex multigenic disease. Here we show that two N-terminal cadherin-like domains, CLD1 and CLD2 (CLD(1-2)), from human RET adopt a clam-shell arrangement distinct from that of classical cadherins. CLD1 structural elements and disulfide composition are unique to mammals, indicating an unexpected structural diversity within higher and lower vertebrate RET CLD regions. We identify two unpaired cysteines that predispose human RET to maturation impediments in the endoplasmic reticulum and establish a quantitative cell-based RET maturation assay that offers a biochemical correlate of HSCR disease severity. Our findings provide a key conceptual framework and means of testing and predicting genotype-phenotype correlations in HSCR.

Involvement of heterogeneous ribonucleoprotein F in the regulation of cell proliferation via the mammalian target of rapamycin/S6 kinase 2 pathway.

The S6 kinases (S6Ks) have been linked to a number of cellular processes, including translation, insulin metabolism, cell survival, and RNA splicing. Signaling via the phosphotidylinositol 3-kinase and mammalian target of rapamycin (mTOR) pathways is critical in regulating the activity and subcellular localization of S6Ks. To date, nuclear functions of both S6K isoforms, S6K1 and S6K2, are not well understood. To better understand S6K nuclear roles, we employed affinity purification of S6Ks from nuclear preparations followed by mass spectrometry analysis for the identification of novel binding partners. In this study, we report that in contrast to S6K1, the S6K2 isoform specifically associates with a number of RNA-binding proteins, including heterogeneous ribonucleoproteins (hnRNPs). We focused on studying the mechanism and physiological relevance of the S6K2 interaction with hnRNP F/H. Interestingly, the S6K2-hnRNP F/H interaction was not affected by mitogenic stimulation, whereas mTOR binding to hnRNP F/H was induced by serum stimulation. In addition, we define a new role of hnRNP F in driving cell proliferation, which could be partially attenuated by rapamycin treatment. S6K2-driven cell proliferation, on the other hand, could be blocked by small interfering RNA-mediated down-regulation of hnRNP F. These results demonstrate that the specific interaction between mTOR and S6K2 with hnRNPs is implicated in the regulation of cell proliferation.

The identification and characterization of novel PKCepsilon phosphorylation sites provide evidence for functional cross-talk within the PKC superfamily.

PKCepsilon (protein kinase Cepsilon) is a phospholipid-dependent serine/threonine kinase that has been implicated in a broad array of cellular processes, including proliferation, survival, migration, invasion and transformation. Here we demonstrate that, in vitro, PKCepsilon undergoes autophosphorylation at three novel sites, Ser(234), Ser(316) and Ser(368), each of which is unique to this PKC isoform and is evolutionarily conserved. We show that these sites are phosphorylated over a range of mammalian cell lines in response to a number of different stimuli. Unexpectedly, we find that, in a cellular context, these phosphorylation events can be mediated in-trans by cPKC (classical PKC) isoforms. The functional significance of this cross-talk is illustrated through the observation that the cPKC-mediated phosphorylation of PKCepsilon at residue Ser(368) controls an established PKCepsilon scaffold interaction. Thus our current findings identify three new phosphorylation sites that contribute to the isoform-specific function of PKCepsilon and highlight a novel and direct means of cross-talk between different members of the PKC superfamily.

Novel phosphorylation site markers of protein kinase C delta activation.

Protein kinase C delta (PKCdelta) is a Ser/Thr kinase which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. Here, we demonstrate that PKCdelta undergoes in vitro autophosphorylation at three sites within its V3 region (S299, S302, S304), each of which is unique to this PKC isoform and evolutionarily conserved. We demonstrate that S299 and S304 can be phosphorylated in mammalian cells following phorbol ester stimulation and that S299-phosphorylated PKCdelta is localised to both the plasma and nuclear membranes. These data indicate that PKCdelta is phosphorylated upon activation and that phospho-S299 represents a useful marker of the activated enzyme.

Labeling and identification of LNCaP cell surface proteins: a pilot study.

BACKGROUND: Membrane proteins provide the interface between the cell and its environment and are responsible for cell adhesion, mobility, and intracellular signaling. Previous studies have focused on the LNCaP whole cell proteome and transcriptome but little is known about proteins at the prostate cell membrane and how they change in response to androgens. MATERIALS AND METHODS: Following treatment with R1881 or vehicle, membrane proteins of the prostate cancer LNCaP cell line were tagged with biotin using EZ-link sulfo-NHS-LC-biotin. Using the tag membrane proteins were purified and separated using two-dimensional gel electrophoresis and identified using mass spectrometry. E-cadherin and low density lipoprotein receptor (LDLR) were used as positive controls and also investigated following bicalutamide treatment. Membrane localization and androgen-regulation of proteins was confirmed using sub-cellular fractionation, Western blotting and microscopy. RESULTS: We have demonstrated efficient and specific protein biotinylation and purification of LNCaP plasma membrane proteins using Western analysis. E-cadherin and LDLR were regulated at the cell surface in response to R1881 and bicalutamide. Mass spectrometry identified several androgen-regulated membrane associated proteins including Prx-3 and GRP78 which are known to localize to other cellular compartments as well as the plasma membrane. We confirmed the localization of the identified proteins in LNCaP cells by co-localization with E-cadherin and immunohistochemistry of prostate tissue. CONCLUSION: Cell surface biotinylation is an effective technique for identifying membrane proteins in the LNCaP prostate cancer cell line. We have demonstrated the identification of androgen-regulated membrane proteins and their validation in tissue samples.

PLCgamma is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly.

Nuclear envelope assembly is an essential event in each cell cycle but the proteins and lipids involved in its regulation remain mostly unknown. Assembly involves membrane fusions but neither specific SNAREs nor Rab GTPases have been identified in its control. We report that a precursor membrane population (MV1) required for NE assembly has a unique lipid composition consisting prominently of poly-phosphatidylinositides. The lipid composition was determined by adapting HPLC electrospray ionisation tandem mass spectrometry to phosphoinositide analysis, revealing the capacity of this technique to document dynamic lipid transitions of functional importance in natural membrane populations. MV1 is >100-fold enriched in endogenous PLCgamma and >25-fold enriched in the PLC substrate phosphatidylinositol bisphosphate (PtdInsP2) compared to the second membrane population, derived largely from endoplasmic reticulum (ER), that contributes most of the NE. During NE formation PLCgamma becomes transiently phosphorylated at the tyrosine 783 site indicative of its activation. In addition specific inhibition of PLCgamma blocks nuclear envelope formation. In vivo, PLCgamma is concentrated on vesicles of similar size to purified MV1. These associate with nuclei during the period of NE formation and are distinct from ER membranes. The unprecedented concentration of PLCgamma and its substrate PtdInsP2 in a subset of membranes that binds to only two regions of the nucleus, and activation of PLCgamma by GTP during initial stages of NE formation provide a mechanism for temporal control of NE assembly and offer an explanation for how such a process of membrane fusion can be spatially regulated.

Proteomic identification of a role for the von Hippel Lindau tumour suppressor in changes in the expression of mitochondrial proteins and septin 2 in renal cell carcinoma.

The von Hippel Lindau (VHL) tumour suppressor gene, VHL, plays a central role in development of sporadic conventional renal cell carcinomas (RCCs). Studying VHL function may, therefore, increase understanding of the pathogenesis of RCC and identify markers/therapeutic targets. Comparison of 2-DE protein profiles of VHL-defective RCC cells (UMRC2) transfected with control vector or wild-type VHL showed differences in 30 proteins, including several novel changes. One of the findings confirmed by Western blotting was up-regulation of the mitochondrial protein ubiquinol cytochrome c reductase complex core protein 2 following VHL transfection, a change that was also observed in two other cell line backgrounds. A marked decrease in expression of this and several other mitochondrial proteins was demonstrated in RCC tissues and using VHL-transfectants, several were shown to exhibit VHL-dependent regulation. Thus, VHL may contribute to the decreased mitochondrial function seen in RCC. A form of septin 2 down-regulated following VHL transfection was also identified. Septin 2 was up-regulated in 12/16 RCCs, while alteration of the form present was also observed in 1/3 tumours analysed. Thus, increased expression of septin 2 is a common event in RCC and protein modification may also alter septin 2 function in a subset of tumours.

Proteomic analysis of primary cell lines identifies protein changes present in renal cell carcinoma.

New markers/targets for renal cell carcinoma (RCC) are needed to enable earlier detection and monitoring of disease and therapeutic targeting. To identify such molecules, normal and RCC-derived primary cell lines have been used as a simplified model system for studying changes that accompany tumorigenesis. Short-term cultures allow enrichment of relevant cell types from tissue samples, which is balanced against the potential for in vitro changes. Examination of 3 proteins with altered expression in RCC tissue showed the maintenance of normal-tumour differences in culture, although some changes were apparent, including alteration in the isoform of aldolase. Comparative analysis of primary cell lines by 2-DE found 43 proteins up-regulated and 29 down-regulated in at least three out of five tumour cell lines. Many of the observed changes have been previously reported in RCC, including up-regulation of several glycolytic enzymes, vimentin and heat shock protein 27, validating the approach. Additionally, several novel changes in protein expression were found, including up-regulation of several proteins involved in actin cytoskeleton organisation such as radixin and moesin, two members of the septin family, and the actin bundling protein, fascin. Validation studies using Western blotting and immunohistochemistry indicate that several of these molecules may be useful as markers for RCC.

RhoE function is regulated by ROCK I-mediated phosphorylation.

The Rho GTPase family member RhoE regulates actin filaments partly by binding to and inhibiting ROCK I, a serine/threonine kinase that induces actomyosin contractility. Here, we show that ROCK I can phosphorylate multiple residues on RhoE in vitro. In cells, ROCK I-phosphorylated RhoE localizes in the cytosol, whereas unphosphorylated RhoE is primarily associated with membranes. Phosphorylation has no effect on RhoE binding to ROCK I, but instead increases RhoE protein stability. Using phospho-specific antibodies, we show that ROCK phosphorylates endogenous RhoE at serine 11 upon cell stimulation with platelet-derived growth factor, and that this phosphorylation requires an active protein kinase C signalling pathway. In addition, we demonstrate that phosphorylation of RhoE correlates with its activity in inducing stress fibre disruption and inhibiting Ras-induced transformation. This is the first demonstration of an endogenous Rho family member being phosphorylated in vivo and indicates that phosphorylation is an important mechanism to control the stability and function of this GTPase-deficient Rho protein.

Androgen receptor is targeted to distinct subcellular compartments in response to different therapeutic antiandrogens.

PURPOSE: Antiandrogens are routinely used in the treatment of prostate cancer. Although they are known to prevent activation of the androgen receptor (AR), little is known about the mechanisms involved. This report represents the first study of the localization of wild-type AR following expression at physiologic relevant levels in prostate cells and treatment with androgen and antiandrogens. EXPERIMENTAL DESIGN: We have characterized a cellular model for prostate cancer using in situ cellular fractionation, proteomics, and confocal microscopy and investigated the effect of antiandrogens in clinical use on the subcellular localization of the AR. RESULTS: Different antiandrogens have diverse effects on the subcellular localization of the AR. Treatment with androgen results in translocation from the cytoplasm to the nucleoplasm, whereas the antiandrogens hydroxyflutamide and bicalutamide lead to reversible association with the nuclear matrix. In contrast, treatment with the antiandrogen cyproterone acetate results in AR association with cytoplasmic membranes and irreversible retention within the cytoplasm. In addition, we demonstrate that AR translocation requires ATP and the cytoskeleton, regardless of ligand. CONCLUSIONS: These results reveal that not all antiandrogens work via the same mechanism and suggest that an informed sequential treatment regime may benefit prostate cancer patients. The observed subnuclear and subcytoplasmic associations of the AR suggest new areas of study to investigate the role of the AR in the response and resistance of prostate cancer to antiandrogen therapy.

Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy. Further exploration into the protein composition of exosomes may increase our understanding of their potential roles in vivo and this study has examined the proteome of exosomes purified from cell supernatants of the melanoma cell lines MeWo and SK-MEL-28. The vesicular nature and size (30-100 nm) of the purified exosomes was confirmed by electron microscopy and sucrose density gradient centrifugation. Western blotting demonstrated the absence of calnexin and cytochrome c, verifying the purity of the exosome preparations, as well as enrichment of MHC class I and the tumour-associated antigens Mart-1 and Mel-CAM. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein profiles of exosomes from the two cell lines were highly comparable and strikingly different from the profiles of the total cell lysates. Mass spectrometric sequencing identified proteins present in 49 protein spots in the exosome lysates. Several of these have been identified previously in exosomes but some are novel, including p120 catenin, radixin, and immunoglobulin superfamily member 8 (PGRL). Proteins present in whole-cell lysates that were significantly reduced or excluded from exosomes were also identified and included several mitochondrial and lysosomal proteins, again confirming the proposed endosomal origin of exosomes. This study presents a starting point for future more in-depth protein studies of tumour-derived exosomes which will aid the understanding of their biogenesis and targeting for use in anti-tumour immunotherapy protocols.

Identification of proteins regulated by interferon-alpha in resistant and sensitive malignant melanoma cell lines.

Treatment of patients with malignant melanoma with interferon-alpha achieves a response in a small but significant subset of patients. Currently, although much is known about interferon biology, little is known about either the particular mechanisms of interferon-alpha activity that are crucial for response or why only some patients respond to interferon-alpha therapy. Two melanoma cell lines (MeWo and MM418) that are known to differ in their response to the antiproliferative activity of interferon-alpha, have been used as a model system to investigate interferon-alpha action. Using a proteomics approach based on two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, several proteins induced in response to interferon-alpha have been identified. These include a number of gene products previously known to be type I interferon responsive (tryptophanyl tRNA synthetase, leucine aminopeptidase, ubiquitin cross-reactive protein, gelsolin, FUSE binding protein 2 and hPNPase) as well as a number of proteins not previously reported to be induced by type I interferon (cathepsin B, proteasomal activator 28alpha and alpha-SNAP). Although the proteins upregulated by interferon-alpha were common between the cell lines when examined at the level of Western blotting, the disparity in the basal level of cathepsin B was striking, raising the possibility that the higher level in MM418 may contribute to the sensitivity of this cell line to interferon-alpha treatment.

Identification of pro-interleukin 16 as a novel target of MAP kinases in activated T lymphocytes.

T lymphocyte activation is controlled by a coordinated web of tyrosine and serine kinases. There is a large body of information about tyrosine kinase substrates in T cells but analysis of serine kinase substrates has been more difficult. Recently we described an antiserum that recognizes serine-phosphorylated peptides corresponding to the substrate sequences for AGC serine kinases. This antiserum, termed PAP-1 (phospho antibody for proteomics-1), has proven useful for probing the serine phosphoproteome of antigen receptor-activated T lymphocytes. The present study shows that PAP-1 can also be used to explore serine kinases activated by cytokines and chemokines in T cells. Using PAP-1, together with proteomic analysis, the precursor form of the cytokine IL-16 (ProIL-16) was shown to be phosphorylated on Ser144 in antigen receptor-, SDF1alpha- and IL-2-activated T cells. Genetic and pharmacological-inhibitor experiments showed that the phosphorylation of ProIL-16 is dependent on activation of the kinases Erk1/2. IL-16 is secreted by mitogen-activated T cells, and the biochemical link between ProIL-16 and Erk1/2, revealed by studies with PAP-1, prompted analysis of the role of MAP kinases in this response. We show that TCR-mediated secretion of IL-16 is dependent on MAP kinases. The present study thus reveals how phosphoproteomic analysis opens previously unrecognized avenues for research, and yields novel insights about targets for MAP kinases in T lymphocytes.

Proteomic changes in renal cancer and co-ordinate demonstration of both the glycolytic and mitochondrial aspects of the Warburg effect.

Renal cell carcinoma (RCC) is the tenth most common cancer although the incidence is increasing. The main clinical problems stem from the relatively late presentation of many patients due to the often asymptomatic nature of the illness, and the relative insensitivity of metastatic disease to conventional chemotherapy and radiotherapy. Despite increasing knowledge of some of the genetic changes underlying sporadic renal cancer such as those involving the Von Hippel Lindau (VHL) gene, many of the underlying pathophysiological changes are ill-defined and there remains a need for the identification of disease markers for use in diagnosis and prognosis or as potential therapeutic targets. This study has used a proteomic approach, based on two-dimensional gel electrophoresis and mass spectrometry, to compare the protein profiles of conventional RCC tissue with patient-matched normal kidney cortex. Sequencing of 32 protein spots with significantly increased expression in RCC samples (>/= 4/6 patients) and 41 proteins whose levels decreased (6/6 patients) confirmed several previously known RCC-associated changes such as increases in Mn-superoxide dismutase, lactate dehydrogenase-A, aldolase A and C, pyruvate kinase M2, and thymidine phosphorylase. Additionally, several previously unknown changes were identified, including increased expression of three members of the annexin family and increased levels of the actin depolymerisation factor cofilin. The Warburg effect was also demonstrated with the identification of increases in proteins involved in the majority of steps in the glycolytic pathway and decreases in the gluconeogenic reactions, together with a parallel decrease in several mitochondrial enzymes. A number of the alterations seen were further confirmed in additional samples by immunohistochemistry, Western blotting, and laser capture microdissection.

Approaches to define antigen receptor-induced serine kinase signal transduction pathways.

In the present report we describe the properties of a novel phospho-specific antiserum that has opened a route to the characterization of antigen receptor-activated serine kinase pathways in lymphocytes. The basis for the present work was that Ser-21 in glycogen synthase kinase 3alpha is robustly phosphorylated following antigen receptor triggering. We predicted accordingly that antigen receptors would also stimulate phosphorylation of other proteins with a similar sequence. To test this idea we raised an antibody against the phospho-peptide RARTSpSFAEP, where pS is a phospho-serine corresponding to the glycogen synthase kinase 3alpha Ser-21 sequence. The resulting antiserum was called phospho antibody for proteomics-1 (PAP-1). The present study describes the properties of PAP-1 and shows that it can reveal quite striking differences in the phospho-proteome of different cell types and is able to pinpoint new targets in important signal transduction pathways. PAP-1 was used to map protein phosphorylations regulated by the antigen receptor in T cells. One of these PAP-1-reactive proteins was purified and revealed to be a previously unrecognized target for antigen receptor signal transduction, namely an "orphan" adapter SLY (Src homology 3 (SH3) domain-containing protein expressed in lymphocytes). The use of sera detecting specific phosphorylation sites is thus proved as a powerful method for the discovery of novel downstream components of antigen receptor signals in T cells.

Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY.

Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. We have performed a detailed analysis of the mouse Hoxb4 intron enhancer to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. As these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site.

Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue preparation and sample limitations.

Laser capture microdissection (LCM) is now well established as a tool for facilitating the enrichment of cells of interest from tissue sections, overcoming the problem of tissue heterogeneity. LCM has been used extensively in combination with analysis at the DNA and RNA levels, but only a small number of studies have employed LCM with subsequent protein analysis, albeit with promising results. This study focuses on the potential of LCM in combination with two-dimensional polyacrylamide gel electrophoresis. The effects of tissue section preparation and sample type were evaluated to fully determine the suitability of using LCM in global protein profiling. The effects of several histochemical stains (hematoxylin and eosin, methyl green and toluidine blue) and immunolabeling on subsequent two-dimensional polyacrylamide gel electrophoresis were investigated. Quantitative analysis was performed to establish the extent of changes in the relative intensity of protein species and their reproducibility. All staining protocols tested were found to be compatible with protein analysis although there was variation in protein recovery and the quality of the protein profiles obtained. LCM of renal and cervix samples indicated that protein yield after dissection was acceptable, although the extent of enrichment and dissection time was tissue-dependent, which may preclude the use of this approach with some tissue types. These results indicate that LCM has potential as a tool in proteomic research.