Proteomic analysis of laser-microdissected paraffin-embedded tissues: (1) Stage-related protein candidates upon non-metastatic lung adenocarcinoma

We used formalin-fixed paraffin-embedded (FFPE) materials for biomarker discovery in cases of lung cancer using proteomic analysis. We conducted a retrospective global proteomic study in order to characterize protein expression reflecting clinical stages of individual patients with stage I lung adenocarcinoma without lymph node involvement (n = 7). In addition, we studied more advanced stage IIIA with spread to lymph nodes (n = 6), because the degree of lymph node involvement is the most important factor for staging. FFPE sections of cancerous lesions resected surgically from patients with well-characterized clinical history were subjected to laser microdissection (LMD) followed by Liquid Tissue? solubilization and digestion trypsin. Spectral counting was used to measure the amounts of proteins identified by shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS). More than 500 proteins were identified from IA and IIIA cases, and non-parametric statistics showed that 81 proteins correlated significantly with stage IA or IIIA. A subset of those proteins were verified by multiple-reaction monitoring mass spectrometric quantitation (MRM assay), described in other paper in this issue. These results demonstrated the technical feasibility of a global proteomic study using clinically well documented FFPE sections, and its possible utility for detailed retrospective disease analyses in order to improve therapeutic strategy.     

Alterations in cellular proteome and secretome upon differentiation from monocyte to macrophage by treatment with phorbol myristate acetate: Insights into biological processes

Monocyte and macrophage are mainly involved in immune response and inflammatory processes. Monocytes circulate in the bloodstream and migrate to various tissues where they can differentiate to macrophages. However, the molecular basis of biological processes involved in this cellular differentiation remains ambiguous. This study was to investigate alterations in cellular and secreted proteins after this differentiation phase. Macrophage was differentiated from U937 human monocytic cell line by treatment with 100 ng/ml phorbol myristate acetate (PMA) for 48 h. Cellular and secreted proteins extracted from PMA-treated cells (macrophages) were compared with those of untreated cells (monocytes) using 2-DE (n = 5 gels/condition; stained with Deep Purple fluorescence dye). Quantitative intensity analysis revealed 81 and 67 protein spots whose levels were significantly altered in cellular proteome and secretome. These proteins were subsequently identified by Q-TOF MS and/or MS/MS analyses. The altered levels of cellular elongation factor-2 (EF-2) and secreted α-tubulin were confirmed by Western blot analysis. Global protein network analysis demonstrated that these altered proteins were involved in cell death, lipid metabolism, cell morphology, cellular movement, and protein folding. Our data may provide some insights into molecular mechanisms of biological processes upon differentiation from monocytes to macrophages.     

Proteomic characterization of the released/secreted proteins of Leishmania (Viannia) braziliensis promastigotes

Extracellular proteins secreted/released by protozoan parasites are key mediators of the host–parasite interaction. To characterise the profile of proteins secreted/released by Leishmania (Viannia) braziliensis promastigotes, a proteomic approach combining two-dimensional electrophoresis (2DE), tandem matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometry, and data mining was carried out. The 2DE map revealed a set of 270 secreted protein spots from which 42 were confidently identified and classified into 11 categories according to Gene Ontology (GeneDB database) and KEEG Ontology annotation of biological processes. Parasite promastigotes were able to secrete/release proteins involved in immunomodulation, signal transduction, and intracellular survival, such as HSP70, acid phosphatase, activated protein kinase C receptor (LACK), elongation factor 1β, and tryparedoxin peroxidase. Data mining showed that ~ 5% of identified proteins present a classical secretion signal whereas ~ 57% were secreted following non-classical secretion mechanisms, indicating that protein export in this primitive eukaryote might proceed mainly by unconventional pathways. This study reports a suitable approach to identify secreted proteins in the culture supernatant of L. braziliensis and provides new perspectives for the study of molecules potentially involved in the early stages of infection.     

Different expression of Fibrinopeptide A and related fragments in serum of type 1 diabetic patients with nephropathy

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is an autoimmune disease affecting about 0.12% of the world's population. Diabetic nephropathy (DN) is a major long-term complication of both types of diabetes and retains a high human, social and economic cost. Thus, the identification of markers for the early detection of DN represents a relevant target of diabetic research.The present work is a pilot study focused on proteomic analysis of serum of controls (n = 9), IDDM patients (n = 10) and DN patients (n = 4) by the ClinProt profiling technology based on mass spectrometry. This approach allowed to identify a pattern of peptides able to differentiate the studied populations with sensitivity and specificity close to 100%. Variance of the results allowed to estimate the sample size needed to keep the expected False Discovery Rate low. Moreover, three peptides differentially expressed in the serum of patients as compared to controls were identified by LC-ESI MS/MS as the whole fibrinopeptide A peptide and two of its fragments, respectively. The two fragments were under-expressed in diabetic patients, while Fibrinopeptide A was over-expressed, suggesting that anomalous turnover of Fibrinopeptide A could be involved in the pathogenesis of DN.     

Proteomic profiles of soluble proteins from the esophageal gland in female Meloidogyne incognita

Meloidogyne incognita can infect multiple plant species. Proteins synthesized in the esophageal glands and secreted through the stylet of plant parasitic nematodes play critical roles in the plant-nematode interactions. Female M. incognita live for approximately 15 days, embedded in a host plant, but their esophageal gland proteins have not yet been comprehensively analyzed. In this study, a new bacterium-contamination-resistant method for collecting soluble proteins from esophageal gland cells (SPEGC) of female M. incognita was established. Approximately 5 μg of freeze-dried proteins could be extracted from 150 female M. incognita. Bands of a one-dimensional SDS–polyacrylamide gel were excised after electrophoresis of 20 μg of protein and were analyzed. Two hundred and forty-six proteins from SPEGC of female M. incognita were identified by LC–MS/MS. Gene Ontology analysis suggests that many of the secreted proteins are involved in protein or carbohydrate metabolism and proteolysis. Some of the SPEGC (46.3%) were predicted to be secreted through classical or non-classical secretory pathways. The described method presents a new approach for the identification of proteins stored in SPEGC of an important plant parasitic nematode. This global proteomic profile of SPEGC provides a basis for future studies to elucidate the functions of proteins secreted from female M. incognita on plant responses.     

Proteomic analysis in mammary glands of rat offspring exposed in utero to bisphenol A

Bisphenol A (BPA) is a ubiquitous environmental contaminant with established endocrine disruptor properties. The objective of our study was to determine the effects of prenatal exposure to BPA on the rat mammary gland proteome in postnatal rats as a first step toward the investigation of translational biomarkers of susceptibility in the human population. Pregnant rats were treated orally with 0, 25 or 250 µg BPA/kg body weight from days 10 to 21 post-conception. Female offspring were euthanized at 21 and 50 days, and mammary glands were collected. Proteomic analysis was conducted using 2-DE, followed by a combination of MALDI-TOF–TOF and LC–MS/MS, which led to the identification of 21 differentially abundant proteins including vimentin, SPARC and 14–3–3. Western blot analysis of key downstream signaling proteins demonstrated increased phospho-AKT, c-Raf, phospho-ERKs-1 and 2, but decreased TGF-β in mammary glands of 50 day old rats exposed prenatally to BPA. Our studies indicate for the first time that key proteins involved in signaling pathways such as cellular proliferation are regulated at the protein level by BPA. This data is expected to aid in the understanding of how BPA may be influencing the susceptibility of the mammary gland to cancer transformation.     

Standardized sample preparation phases for a quantitative measurement of plasma peptidome profiling by MALDI-TOF

A growing body of literature defines MALDI-TOF MS as a technique for studying plasma and serum, thus enabling the detection of proteins, and the generation of reproducible protein profile mass spectra, potentially able to discriminate correctly different biological systems.In this work, the different steps of the pre-analytical phase that may affect the reproducibility of plasma proteome analysis have been carefully considered.The results showed that the method is highly accurate (9.1%) and precise (8.9%) and the calibration curve for the ACTH (18–39), in human plasma, gave a good correlation coefficient (r > 0.99 and r² > 0.98). The limit of detection (LOD) and the limit of quantification (LOQ), relative intensity, were of 0.5 × 10^?9 M and 1.0 × 10^?9 M respectively.Thus, an assay has been developed for the detection of low-abundant and low molecular weight proteins, from human plasma, aiming at the identification of new potential biomarkers. The method was tested on plasma from patients with a first diagnosis of pelvic mass. Statistical analysis of plasma profile generated a sub-profile of 17 peptides with their relative abundance able to discriminate patients bearing malignant or benign tumors. The sensitivity and specificity were 85.7% and 80.0% respectively.     

Metabolomic analysis identifies altered metabolic pathways in Multiple Sclerosis

Multiple sclerosis (MS) is a chronic, demyelinating disease that affects the central nervous system and is characterized by a complex pathogenesis and difficult management. The identification of new biomarkers would be clinically useful for more accurate diagnoses and disease monitoring. Metabolomics, the identification of small endogenous molecules, offers an instantaneous molecular snapshot of the MS phenotype. Here the metabolomic profiles (utilizing plasma from patients with MS) were characterized with a Gas cromatography-mass spectrometry-based platform followed by a multivariate statistical analysis and comparison with a healthy control (HC) population. The obtained partial least square discriminant analysis (PLS-DA) model identified and validated significant metabolic differences between individuals with MS and HC (R2X = 0.223, R2Y = 0.82, Q2 = 0.562; p < 0.001). Among discriminant metabolites phosphate, fructose, myo-inositol, pyroglutamate, threonate, l-leucine, l-asparagine, l-ornithine, l-glutamine, and l-glutamate were correctly identified, and some resulted as unknown. A receiver operating characteristic (ROC) curve with AUC 0.84 (p = 0.01; CI: 0.75–1) generated with the concentrations of the discriminant metabolites, supported the strength of the model. Pathway analysis indicated asparagine and citrulline biosynthesis as the main canonical pathways involved in MS. Changes in the citrulline biosynthesis pathway suggests the involvement of oxidative stress during neuronal damage. The results confirmed metabolomics as a useful approach to better understand the pathogenesis of MS and to provide new biomarkers for the disease to be used together with clinical data.     

Production of volatile organic compounds by Aureobasidium pullulans as a potential mechanism of action against postharvest fruit pathogens

Two Aureobasidium pullulans strains (L1 and L8), effective against some fruit postharvest pathogens were evaluated for VOCs production as a part of their modes of action towards five pathogens (Botrytis cinerea, Colletotrichum acutatum, Penicillium expansum, Penicillium digitatum and Penicillium italicum). The VOCs were assayed with a double petri dish assay against conidia germination of target pathogens. Results obtained showed that the VOCs generated by the antagonists inhibited significantly the conidia germination of all pathogens compared to the control. In particular, the conidia germination of all Penicillium was completely inhibited by VOCs produced by L1 and L8. In in vivo tests, apples and oranges were artificially inoculated with pathogen conidia and then biofumigated with VOCs emitted by both antagonists. The antagonistic treatment controlled significantly pathogen infection, confirming the results obtained in vitro tests. The best L1 and L8 VOCs activity was observed on apple inoculated with B. cinerea where the lesion diameter reduction observed was greater than the 88%. The compounds emitted by L1 and L8 strains were identified with the solid-phase microextraction (SPME)–gas chromatographic technique. Compounds as 2-phenyl, 1-butanol-3-methyl, 1-butanol-2-methyl and 1-propanol-2-methyl belonging to the group of alcohols were mainly produced for both strains, in the first 96 h of growth. These compounds were confirmed by comparison with standards. The pure compounds of VOCs cited above were used to determine the EC₅₀ values for conidia germination of pathogens. The 1-propanol-2-methyl was the VOC least active against all tested fungi, with the EC₅₀ values over 0.8 μl ml⁻¹, while the 2-phenethyl alcohol was the most active with EC₅₀ values lower than 0.8 μl ml⁻¹, except for the C. acutatum (1.97 μl ml⁻¹). The present study demonstrated, for the first time, that the production of VOCs could play an essential role in the antagonistic activity of two A. pullulans strains against five fruit postharvest pathogens.     

Effect of crude oil petroleum hydrocarbons on protein expression of the prawn Macrobrachium borellii

Hydrocarbon pollution is a major environmental threat to ecosystems in marine and freshwater environments, but its toxicological effect on aquatic organisms remains little studied. A proteomic approach was used to analyze the effect of a freshwater oil spill on the prawn Macrobrachium borellii. To this aim, proteins were extracted from midgut gland (hepatopancreas) of male and female prawns exposed 7 days to a sublethal concentration (0.6 ppm) of water-soluble fraction of crude oil (WSF). Exposure to WSF induced responses at the protein expression level. Two-dimensional gel electrophoresis (2-DE) revealed 10 protein spots that were differentially expressed by WSF exposure. Seven proteins were identified using MS/MS and de novo sequencing. Nm23 oncoprotein, arginine methyltransferase, fatty aldehyde dehydrogenase and glutathione S-transferase were down-regulated, whereas two glyceraldehyde-3-phosphate dehydrogenase isoforms and a lipocalin-like crustacyanin (CTC) were up-regulated after WSF exposure. CTC mRNA levels were further analyzed by quantitative real-time PCR showing an increased expression after WSF exposure. The proteins identified are involved in carbohydrate and amino acid metabolism, detoxification, transport of hydrophobic molecules and cellular homeostasis among others. These results provide evidence for better understanding the toxic mechanisms of hydrocarbons. Moreover, some of these differentially expressed proteins would be employed as potential novel biomarkers.     

Label-free quantitative mass spectrometry from formalin-fixed paraffin-embedded samples of nasopharyngeal carcinoma: Preliminary results from a non-endemic European cohort of patients

AimReport our results of biomarker discovery in formalin-fixed paraffin-embedded (FFPE) nasopharyngeal carcinoma (NPC) via proteomic analysis.BackgroundNasopharyngeal carcinoma (NPC) is a rare cancer in Western countries. Proteomic analysis have already been reported as a useful tool to provide biomarkers. Formalin-fixed paraffin-embedded (FFPE) samples, despite largely underused, can provide invaluable information for biomarker research via proteomic analysis.MethodsFFPE samples of NPC were submitted to protein extraction followed by FASP-digestion and label-free quantitative mass spectrometry (MS). Patients’ received concurrent chemoradiation with or without adjuvant chemotherapy as per Intergroup 0099 trial. IMRT was delivered following the RTOG0615 specifications. Toxicity was scored using the CTCAE 4.03 tables. Survival was estimated using Kaplan–Meier curves. Log-rank was used to detect differences. KEGG ontology graphics were generated.Results28 FFPE samples from NPC patients were used. Patients were: 79% male, 97% Caucasians, 86% WHO type 3, 40% T1, 10% T2, 25% T3, and 25% T4. With a median follow up of 37 months, local control was 83 (T1, 100% T2, T3 and T4), overall survival was 84%, and six patients developed distant metastases. All five patients that died were due to metastatic disease. Tumor samples contained a median of 75% of tumor material. We found Epstein–Barr (EBV) and Herpes simplex (HSV) viruses’ related proteins significantly present in early-stage primary NPC (T1 and T2, p < 0.01). A pool of 10 proteins was statistically up-regulated in the metastatic group of patients (p < 0.01). Median survival from this M1 group was <1 year (p < 0.001).ConclusionsFFPE samples yielded adequate material for MS analysis. We found EBV and HSV related proteins on early-stage NPC, and proteomic profiling associated with distant metastases, potential candidates of disease biomarkers. Validation is needed.     

Molecular ecological responses of the dinoflagellate Karenia mikimotoi to phosphate stress

Karenia mikimotoi is a toxic, widespread dinoflagellate which could produce hemolytic toxins and ichthyotoxins affecting fisheries within the area of its bloom. Previous ecophysiological studies indicated that the enhance of environmental phosphate concentration could promote the growth of K. mikimotoi. Intrinsic mechanisms regarding the effects of external phosphate on its photosynthesis, cell cycle succession and differential proteins’ expressions are still unknown. K. mikimotoi was cultured in phosphate-deprived medium, while the culture in f/2 medium (Guillard, 1975) was introduced as phosphate-sufficient control experiment. Cell counts and phosphate concentration detection were performed every other day. Flowcytometry was applied to measure cell cycle succession and chlorophyll fluorescence intensity fluctuation. Differential proteomics expression was examined by SDS-PAGE tandem LTQ Orbitrap MS/MS spectrometry. Functions of each differential protein were searched within NCBInr protein database and Swissprot database. Our study demonstrated that phosphate stress inhibited growth and cell cycle succession of K. mikimotoi remarkably (p < 0.01). Algal chlorophyll fluorescence intensity was significantly affected by phosphate deprivation (p < 0.05). 11 species of differential proteins were detected only in phosphate-limited culture sample which related to stress signal transduction, vacuolar phosphate release, phospholipid degradation, organic acid synthesis and phagotrophy. 4 kinds of differential proteins were identified only in f/2 medium culture sample which referred to cell proliferation, glycolysis, SAM cycle and polyamine production. Based on analysis of differential proteomic functional annotation, we hypothesized proteomic response mechanism of K. mikimotoi to phosphate stress. Molecular biological responses of dinoflagellate K. mikimotoi to phosphate stress was explored.     

Biocatalytic acylation of sugar alcohols by 3-(4-hydroxyphenyl)propionic acid

Enzymatic synthesis of aromatic esters of four different sugar alcohols (xylitol, arabitol, mannitol, and sorbitol) with 3-(4-hydroxyphenyl)propionic acid was performed in organic solvent medium, using immobilized Candida antarctica lipase (Novozyme 435), and molecular sieves for control of the water content. The influence of reaction parameters on the conversion has been investigated, including reaction time, temperature, alcohol/acid molar ratio, and enzyme amount. The highest conversions (94% for xylitol, 98% for arabitol, 80% for mannitol, and 93% for sorbitol) were obtained in pure tert-butanol at 60 °C and 72 h reaction time, 0.3 alcohol/acid molar ratio, and 0.5 g/mol enzyme/substrate ratio. The isolated new sugar alcohols esters were identified by different spectral analyses. MALDI-TOF MS analysis showed the formation of monoesters, diesters, and small quantities of triesters for all investigated sugar alcohols. The catalytic efficiency of the enzyme was higher for the pentitol substrates, decreasing in the following order: arabitol > xylitol > sorbitol > mannitol. These new compounds could have interesting applications in food, pharmaceutical and cosmetic formulations.     

Wheat quality related differential expressions of albumins and globulins revealed by two-dimensional difference gel electrophoresis (2-D DIGE)

Comparative proteomics analysis offers a new approach to identify differential proteins among different wheat genotypes and developmental stages. In this study, the non-prolamin expression profiles during grain development of two common or bread wheat cultivars (Triticum aestivum L.), Jing 411 and Sunstate, with different quality properties were analyzed using two-dimensional difference gel electrophoresis (2-D DIGE). Five grain developmental stages during the post-anthesis period were sampled corresponding to the cumulative averages of daily temperatures (°C: 156 °C, 250 °C, 354 °C, 447 °C and 749.5 °C). More than 400 differential protein spots detected at one or more of the developmental stages of the two cultivars were monitored, among which 230 proteins were identified by MS. Of the identified proteins, more than 85% were enzymes possessing different physiological functions. A total of 36 differential proteins were characterized between the two varieties, which are likely to be related to wheat quality attributes. About one quarter of the proteins identified expressed in multiple spots with different pIs and molecular masses, implying certain post-translational modifications (PTMs) of proteins such as phosphorylations and glycosylations. The results provide new insights into biochemical mechanisms for grain development and quality.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate aminoflavone (NSC 686288) in human plasma

A reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed and validated for determination of aminoflavone (AF) in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.25 mL of plasma with 1.0 mL ethyl acetate containing 50 ng/mL of the internal standard zileuton. The analytes were separated on a Waters X-Terra? MS C₁₈ column using a mobile phase consisting of methanol/water containing 0.45% formic acid (70:30, v/v) and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the AF concentration range of 5–2000 ng/mL in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL for AF in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully applied to characterize AF plasma concentration-time profile in the cancer patients in a phase I trial.     

Simultaneous analysis of isomers of escin saponins in human plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study after oral administration

A rapid and sensitive bioassay based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous determination of four isomeric escin saponins (escin Ia, escin Ib, isoescin Ia and isoescin Ib) in human plasma has been developed and validated. Sample preparation of plasma after addition of telmisartan as internal standard (I.S.) involved solid-phase extraction (SPE) on C18 cartridges. Separation was based on reversed phase chromatography using gradient elution with methanol–acetonitrile (50:50, v/v) and 10 mM ammonium acetate solution (pH 6.8). MS/MS detection in the positive ion mode used multiple reaction monitoring of the transition at m/z 1113.8 → 807.6. Stability issues with the four saponins required the addition of formic acid to plasma samples prior to storage at ?80 °C and analysis within 30 days. The method was linear at concentrations up to 10 ng/mL with correlation coefficients > 0.996 for all analytes. The lower limit of quantitation (LLOQ) for all four saponins was 33 pg/mL. Intra- and inter-day precisions (as relative standard deviation) were all <15% and accuracies (as relative error) in the range ?5.3% to 6.1%. The method was successfully applied to a pharmacokinetic study of escins in healthy volunteers after oral administration of sodium aescinate tablets containing 60 mg escin saponins.     

Changes induced by two levels of cadmium toxicity in the 2-DE protein profile of tomato roots

Tomato is an important crop from nutritional and economical points of view, and it is grown in greenhouses, where special substrates and the use of recycled water imply an increased risk of Cd accumulation. We investigated tomato root responses to low (10 µM) and high (100 µM) Cd concentrations at the root proteome level. Root extract proteome maps were obtained by 2-DE, and an average of 121, 145 and 93 spots were detected in the 0, 10 and 100 µM Cd treatments, respectively. The low Cd treatment (10 µM) resulted in significant and higher than 2-fold changes in the relative amounts of 36 polypeptides, with 27 of them identified by mass spectrometry, whereas the 100 µM Cd treatment resulted in changes in the relative amounts of 41 polypeptides, with 33 of them being identified. The 2-DE based proteomic approach allowed assessing the main metabolic pathways affected by Cd toxicity. Our results suggests that the 10 µM Cd treatment elicits proteomic responses similar to those observed in Fe deficiency, including activation of the glycolytic pathway, TCA cycle and respiration, whereas the 100 µM Cd treatment responses are more likely due to true Cd toxicity, with a general shutdown of carbon metabolism and increases in stress related and detoxification proteins.     

A comparative study of different dyes for the detection of proteomes derived from Escherichia coli and MDCK cells: Sensitivity and selectivity

We compared sensitivity and selectivity of five dyes for detection of 2D PAGE-resolved proteins derived from Escherichia coli and MDCK cells. The sensitivity of these dyes was in the following order: SYPRO Ruby > Deep Purple > CBB-G250 > CBB-R250 > Colloidal Gold. Also, we report herein for the first time the application of Colloidal Gold (which is commonly used for staining proteins on blotted membranes) for in-gel staining of proteins. For E. coli, most of the dyes preferably detected proteins with pI range of 4.0–6.9, whereas Deep Purple preferably detected proteins with less acidic range (pI 5.0–7.9). For MDCK cells, while other dyes preferably stained proteins at pI range of 5.0–7.9, Colloidal Gold preferably stained more basic proteins (pI 7.0–9.9). This preferential staining property of Colloidal Gold to basic proteins was confirmed in SDS-PAGE-separated lysozyme (pI 9.4), compared to calmodulin (pI 4.0) and albumin (pI 6.0). These data provide useful information to select appropriate dyes for gel-based proteomic analysis of individual samples.     

Heterogeneity in thylakoid membrane proteome of Synechocystis 6803

Cell-free extracts of Synechocystis 6803 were fractionated by successive ultracentrifugation at 40,000 × g, 90,000 × g and 150,000 × g to obtain the three thylakoid fractions designated as 40 k, 90 k and 150 k fractions respectively. These fractions showed differences in absorption and emission spectra. Nano-LC-ESI-Q-TOF MS analysis identified 123 proteins belonging to membrane as well as cytosolic fraction. Out of these proteins, there were 22 proteins with transmembrane helices and 12 proteins with signal peptide. There were 77 proteins common across all the three fractions. Most of these proteins were subunits of photosynthetic complexes, CF₀–CF₁ ATP synthase or ribosomal proteins. Among the rest of the proteins, 8 were exclusive to 40 k fraction, 3 were exclusive to 90 k fraction and 13 were exclusive to 150 k fraction. There were 10 proteins common between 40 k and 90 k fractions and 12 proteins common between 90 k and 150 k fractions. There were no common proteins detected between 40 k and 150 fractions. The results suggested existence of heterogeneity in thylakoids of Synechocystis 6803, which may lead to micro-compartmentation and functional heterogeneity in the thylakoids of this organism as seen previously.